Note: Descriptions are shown in the official language in which they were submitted.
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Genomic sequence of the purH gene and purH-related biallelic markers
FIELD OF THE INVENTION
The invention concerns the genomic and cDNA sequences of the purH gene,
biallelic
markers of the pzrrH gene and the association established between these
markers and prostate
cancer. The invention provides means to determine the predisposition of
individuals to prostate
cancer as well as means for the diagnosis of this cancer and for the
prognosis/detection of an
eventual treatment response to therapeutic agents acting against prostate
cancer.
BACKGROUND OF THE INVENTION
Prostate cancer
The incidence of prostate cancer has dramatically increased over the last
decades. It
averages 30-50/100,000 males in Western European countries as well as within
the US White
male population. In these countries, it has recently become the most commonly
diagnosed
malignancy, being one of every four cancers diagnosed in American males.
Prostate cancer's
incidence is very much population specific, since it varies from 2/100,000 in
China, to over
80/100,000 among African-American males.
In France, the incidence of prostate cancer is 35/100,000 males and it is
increasing by
10/100,000 per decade. Mortality due to prostate cancer is also growing
accordingly. It is the
second cause of cancer death among French males, and the first one among
French males aged
over 70. This makes prostate cancer a serious burden in terms of public
health.
Prostate cancer is a latent disease. Many men carry prostate cancer cells
without overt
signs of disease. Autopsies of individuals dying of other causes show prostate
cancer cells in 30
% of men at age 50 and in 60 % of men at age 80. Furthermore, prostate cancer
can take up to
10 years to kill a patient after the initial diagnosis.
The progression of the disease usually goes from a well-defined mass within
the prostate
to a breakdown and invasion of the lateral marQins of the prostate, followed
by metastasis to
regional lymph nodes, and metastasis to the bone inarrow. Cancer metastasis to
bone is common
and often associated with uncontrollable pain.
Unfortunately, in 80 % of cases, diagnosis of prostate cancer is established
when the
disease has already metastasized to the bones. Of special interest is the
observation that prostate
cancers frequently grow more rapidly in sites of metastasis than within the
prostate itself.
Early-stage diagnosis of prostate cancer mainly relies today on Prostate
Specific Antigen
(PSA) dosage, and allows the detection of prostate cancer seven years before
clinical symptoms
become apparent. The effectiveness of PSA dosage diagnosis is. liowever,
limited due to its
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inability to discriminate between malignant and non-malignant affections of
the organ and
because not all prostate cancers give rise to an elevated serum PSA
concentration. Furthermore,
PSA dosage and other currently available approaches such as physical
examination, tissue
biopsy and bone scans are of limited value in predicting disease progression.
Therefore, there is
a strong need for a reliable diagnostic procedure which would enable a more
systematic early-
stage prostate cancer prognosis.
Although an early-stage prostate cancer prognosis is important, the
possibility of
measuring the period of time during which treatment can be deferred is also
interesting as
currently available medicaments are expensive and generate important adverse
effects.
However, the aggressiveness of prostate tumors varies widely. Some tumors are
relatively
aggressive, doubling every six months whereas others are slow-growing,
doubling once every
five years. In fact, the majority of prostate cancers grows relatively slowly
and are never
clinically manifested. Very often, affected patients are among the elderly and
die from another
disease before prostate cancer actually develops. Thus, a significant question
in treating prostate
carcinoma is how to discriminate betriveen tumors that will progress and those
that will not
progress during the expected lifetime of the patient.
Hence, there is also a strong need for detection means which may be used to
evaluate the
aggressiveness or the development potential of prostate cancer tumors once
diagnosed.
Furthermore, at the present time, there is no means to predict prostate cancer
susceptibility. It would also be very beneficial to detect individual
susceptibility to prostate
cancer. This could allow preventive treatment and a careful follow up of the
development of the
tumor.
A further consequence of the slow growth rate of prostate cancer is that few
cancer cells
are actively dividing at any one time, rendering prostate cancer generally
resistant to radiation
and chemotherapy. Surgery is the mainstay of treatment but it is largely
ineffective and removes
the ejaculatory ducts, resulting in impotence. Oral oestrogens and luteinizing
releasing hormone
analogs are also used for treatment of prostate cancer. These hormonal
treatments provide
marked improvement for many patients, but they only provide temporary relief.
Indeed, most of
these cancers soon relapse with the development of hormone-resistant tumor
cells and the
oestrogen treatment can lead to serious cardiovascular complications.
Consequently, there is a
strong need for preventive and curative treatment of prostate cancer.
Developing reliable means of accessing efficacy and tolerance prognoses could
be of
extreme value in prostate cancer therapy. Indeed. hormonal tlierapy, the main
treatment
currently available, presents important side effects. The use of chemotherapy
is limited because
of the small number of patients with chemosensitive tumors. Furtliermore the
aae profile of the
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prostate cancer patient and intolerance to chemotherapy make the systematic
use of this
treatment very difficult.
Therefore, a valuable assessment of the eventual efficacy of a medicament to
be
administered to a prostate cancer patent as well as the patent's eventual
tolerance to it may
permit to enhance the benefit/risk ratio of prostate cancer treatment.
SUMMARY OF THE INVENTION
purH gene
The purH gene encodes a bifunctional protein which exhibits the final two
activities of
the purine nucleotide biosynthetic pathway, AICARFT and IMPCH (Rayl e al.,
1996; Sugita et
al, 1997). The human gene is located on the long arm of chromosome 2, between
bands q34 and
q35. The human purH cDNA previously described is 1776 base pairs in length
encoding for a
591-amino acid polypeptide. IMPCHase and AICARFT activities are located within
the N-
terminal and C-terminal regions, respectively.
The present invention stems from the isolation and characterization of the
whole
genomic sequence of the purH gene including its regulatory regions.
Oligonucleotide probes
and primers hybridizing specifically with a genomic sequence of purH are also
part of the
invention. A further object of the invention consists of recombinant vectors
comprising any of
the nucleic acid sequences described in the present invention, and in
particular of recombinant
vectors comprising the regulatory region of purH or a sequence encoding the
purH enzyme, as
well as cell hosts comprising said nucleic acid sequences or recombinant
vectors. The invention
also encompasses methods of screening of molecules which modulate or inhibit
the expression
of the purH gene. The invention also comprises a new allelic variant of the
purH protein.
The invention is also directed to biallelic markers that are located within
the purH
genomic sequence or that are in linkage disequilibrium with the purHgene,
these biallelic
markers representing useful tools in order to identify a statistically
significant association
between specific alleles ofpurHgene and diseases such as cancer, more
particularly prostate
cancer. These association methods are within the scope of the invention.
More particularly, the present invention stems from the identification of
genetic
associations between alleles of biallelic markers of the purHgene and cancer,
more particularly
prostate cancer, as confirmed and characterized in a panel of human subjects.
Methods and products are provided for the molecular detection of a genetic
susceptibility to cancer, more particularly prostate cancer, the level of
aggressiveness of cancer,
or prostate cancer tumors, an early onset of cancer, or prostate cancer, a
beneficial response to or
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side effects related to treatment against cancer, or prostate cancer. They can
be used for
diagnosis, staging, prognosis, and monitoring of such a disease, which
processes can be further
included within treatment approaches. The invention also provides for the
efficient design and
evaluation of suitable therapeutic solutions including individualized
strategies for optimizing
drug usage, and screening of potential new medicament candidates.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a table demonstrating the results of a haplotype association
analysis between
sporadic prostate cancer and haplotypes which consist of biallelic markers of
the invention. In
this haplotype analysis, 294 sporadic cases and 3 13 controls were considered.
Figure 2 is a table demonstrating the results of a haplotype association
analysis between
familial prostate cancer and haplotypes which consist of biallelic markers of
the invention. In
this haplotype analysis, 197 familial cases and 313 controls were considered.
Figure 3 is a table demonstrating the results of a haplotype frequency
analysis including
permutation testing.
Figure 4 is a table demonstrating the results of a haplotype association
analysis between
sporadic prostate cancer and haplotypes which consist of biallelic markers of
the purH gene. In
this haplotype analysis, 294 sporadic cases and 313 controls were considered.
Figure 4A
presents the results with the 2-biallelic marker haplotypes and figure 4B
presents the results with
the 3-biallelic marker haplotypes.
Figure 5 is a table demonstrating the haplotype frequency analysis for the
preferred 2-
biallelic marker haplotype comprising biallelic markers of the purH gene.
Figure 6 is a block diagram of an exemplary computer system.
Figure 7 is a flow diagram illustrating one embodiment of a process 200 for
comparing a
new nucleotide or protein sequence with a database of sequences in order to
determine the
homology levels between the new sequence and the sequences in the database.
Figure 8 is a flow diagram illustrating one embodiment of a process 250 in a
computer
for determining whether two sequences are homologous.
Figure 9 is a flow diagram illustrating one embodiment of an identifier
process 300 for
detecting the presence of a feature in a sequence.
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BRIEF DESCRIPTION OF THE SEQUENCES PROVIDED IN THE SEQUENCE
LISTING
SEQ ID No I contains a genomic sequence of pzrrH comprising the 5' regulatory
region
(upstream untranscribed region), the exons and introns, and the 3' regulatory
region
5 (downstream untranscribed region).
SEQ ID No 2 contains a eDNA sequence of purH.
SEQ ID No 3 contains the amino acid sequence encoded by the cDNA of SEQ ID No
2.
SEQ ID Nos 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
and 22,
respectively contain the nucleotide sequence of the amplicons 99-22578, 99-
22580, 99-22585,
99-23437, 99-23440, 99-23442, 99-23444, 99-23451, 99-23452, 99-28437, 99-
32278, 99-5574,
99-5575, 99-5582, 99-5590, 99-5595, 99-5604, 99-5605, and 99-5608, said
amplicons
comprising the non-genic purH-related biallelic markers.
SEQ ID No 23 contains a primer containing the additional PU 5' sequence
described
further in Example 2
SEQ ID No 24 contains a primer containing the additional RP 5' sequence
described
further in Example 2.
In accordance with the regulations relating to Sequence Listings, the
following codes
have been used in the Sequence Listing to indicate the locations of biallelic
markers within the
sequences and to identify each of the alleles present at the polymorphic base.
The code "r" in
the sequences indicates that one allele of the polymorphic base is a guanine,
while the other
allele is an adenine. The code "v" in the sequences indicates that one allele
of the polymorphic
base is a thymine, while the other allele is a cvtosine. The code "m" in the
sequences indicates
that one allele of the polymorphic base is an adenine. while the other allele
is an cytosine. The
code "k" in the sequences indicates that one allele of the polymorphic base is
a guanine, while
the other allele is a thymine. The code "s" in the sequences indicates that
one allele of the
polymorphic base is a guanine, while the other allele is a cytosine. The code
"w" in the
sequences indicates that one allele of the polymorphic base is an adenine,
while the other allele
is an thymine. The nucleotide code of the original allele for each biallelic
marker is the
following:
Biallelic marker OriLyinal allele
99-32284-107 C
99-5602-372 C
5-290-32 C
99-22573-321 C
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99-22586-300 G
99-22586-39 C
99-5596-197 G
5-293-76 C
5-293-155 A
5-294-285 G
99-23454-317 A
99-23454-105 G
99-15528-333 G
99-15798-86 A
5-297-209 A
99-32281-276 C
99-32281-26 T
5-298-376 G
99-23460-199 G
In some instances, the polymorphic bases of the biallelic markers alter the
identity of an
amino acids in the encoded polypeptide. This is indicated in the accompanying
Sequence
Listing by use of the feature VARIANT, placement of an Xaa at the position of
the polymorphic
amino acid, and definition of Xaa as the two alternative amino acids. For
example if one allele of
a biallelic marker is the codon CAC, which encodes histidine, while the other
allele of the
biallelic marker is CAA, which encodes glutamine. the Sequence Listing for the
encoded
polypeptide will contain an Xaa at the location of the polymorphic aniino
acid. In this instance,
Xaa would be defined as being histidine or glutamine.
In other instances, Xaa may indicate an amino acid whose identity is unknown
because
of nucleotide sequence ambiguity. In this instance, the feature UNSURE is
used, placement of an
Xaa at the position of the unknown amino acid and definition of Xaa as being
any of the 20
amino acids or a limited number of amino acids suggested by the genetic code.
DETAILED DESCRIPTION
The present invention provides tiie genomic sequence of the purH gene and
further
provides biallelic markers derived from the patrH locus. The pin-H-related
biallelic markers of
the present invention offer the possibility of rapid, high throughput
genotyping of a large number
of individuals. The biallelic markers of the present invention can be used in
any method of
genetic analysis including linkage studies in families, linkage disequilibrium
studies in
populations and association studies of case-control populations. An important
aspect of the
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present invention is that biallelic markers allow association studies to be
performed to identify
genes involved in complex traits. As part of the present invention an
association between alleles
of purH -related biallelic markers and prostate cancer was established.
Definitions
Before describing the invention in greater detail, the following definitions
are set forth to
illustrate and define the meaning and scope of the terms used to describe the
invention herein.
The term "purH gene", when used herein, encompasses genomic, mRNA and cDNA
sequences encoding the purH protein, including the untranslated regulatory
regions of the
genomic DNA.
The term "heterologous protein", when used herein, is intended to designate
any protein
or polypeptide other than the purH protein. More particularly, the
heterologous protein is a
compound which can be used as a marker in further experiments with a purH
regulatory region.
The term "isolated" requires that the material be removed from its original
environment
(e. g., the natural environment if it is naturally occurring). For example, a
naturally-occurring
polynucleotide or polypeptide present in a living animal is not isolated, but
the same
polynucleotide or DNA or polypeptide, separated from some or all of the
coexisting materials in
the natural system, is isolated. Such polynucleotide could be part of a vector
and/or such
polynucleotide or polypeptide could be part of a composition, and still be
isolated in that the
vector or composition is not part of its natural environment.
The term " urp ified" does not require absolute purity; rather, it is intended
as a relative
definition. Purification of starting material or natural material to at least
one order of magnitude,
preferablv two or three orders, and more preferablv four or five orders of
magnitude is expressly
contemplated. As an example, purification from 0.1 % concentration to 10 %
concentration is
two orders of magnitude. The term "purified" is used herein to describe a
polynucleotide or
polynucleotide vector of the invention which has been separated from other
compounds
including, but not limited to other nucleic acids. carbohydrates, lipids and
proteins (such as the
enzymes used in the synthesis of the polvnucleotide), or the separation of
covalently closed
polynucleotides from linear polynucleotides. A polvnucleotide is substantially
pure when at
least about 50%, preferably 60 to 75% of a sample exhibits a single
polvnucleotide sequence and
conformation (linear versus covalentlv close). A substantially pure
polynucleotide tvpically
comprises about 50%, preferably 60 to 90% weight/weight of a nucleic acid
sample, more
usually about 95%, and preferably is over about 99% pure. Polvnucleotide
purity or
homogeneity is indicated by a number of ineans xvell known in the art, such as
agarose or
polyacrvlamide gel electrophoresis of a sample. followed bv visualizing a
single polvnucleotide
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band upon staining the gel. For certain purposes higher resolution can be
provided by using
HPLC or other means well known in the art.
The term "polypeptide" refers to a polymer of amino acids without regard to
the length
of the polymer; thus, peptides, oligopeptides, and proteins are included
within the definition of
polypeptide. This term also does not specify or exclude post-expression
modifications of
polypeptides, for example, polypeptides which include the covalent attachment
of glycosyl
groups, acetyl groups, phosphate groups, lipid groups and the like are
expressly encompassed by
the term polypeptide. Also included within the definition are polypeptides
which contain one or
more analogs of an amino acid (including, for example, non-naturally occurring
amino acids,
amino acids which only occur naturally in an unrelated biological system,
modified amino acids
from mammalian systems etc.), polypeptides with substituted linkages, as well
as other
modifications known in the art, both naturally occurring and non-naturally
occurring.
The term "recombinant polypeptide" is used herein to refer to polypeptides
that have
been artificially designed and which comprise at least two polypeptide
sequences that are not
found as contiguous polypeptide sequences in their initial natural
environment, or to refer to
polypeptides which have been expressed from a recombinant polynucleotide.
The term " urp ified" is used herein to describe a polypeptide of the
invention which has
been separated from other compounds including, but not limited to nucleic
acids, lipids,
carbohydrates and other proteins. A polypeptide is substantially pure when at
least about 50%,
preferably 60 to 75% of a sample exhibits a single polypeptide sequence. A
substantially pure
polypeptide typically comprises about 50%, preferably 60 to 90% weight/weight
of a protein
sample, more usually about 95%, and preferably is over about 99% pure.
Polypeptide purity or
homogeneity is indicated by a number of means well known in the art, such as
agarose or
polyacrylamide gel electrophoresis of a sample, followed by visualizing a
single polypeptide
band upon staining the gel. For certain purposes higher resolution can be
provided by using
HPLC or other means well known in the art.
As used herein, the term "non-human animal" refers to any non-human
vertebrate, birds
and more usually mammals, preferably primates, farm animals such as swine,
goats, sheep,
donkeys, and horses, rabbits or rodents, more preferably rats or mice. As used
herein, the term
"animal" is used to refer to any vertebrate, preferable a mammal. Both the
terms "animal" and
"mammal" expressly embrace human subjects unless preceded with the term "non-
human".
As used herein, the term "antibody" refers to a polypeptide or group of
polypeptides
which are comprised of at least one binding domain. wliere an antibody binding
domain is
formed from the folding of variable domains of an antibody molecule to form
three-dimensional
5 binding spaces with an internal surface shape and charge distribution
complementary to the
3
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features of an antigenic determinant of an antigen, which allows an
immunological reaction with
the antigen. Antibodies include recombinant proteins comprising the binding
domains, as wells
as fragments, including Fab, Fab', F(ab)2, and F(ab')2 fragments.
As used herein, an "antigenic determinant" is the portion of an antigen
molecule, in this
case a purH polypeptide, that determines the specificity of the antigen-
antibody reaction. An
"epitope" refers to an antigenic determinant of a polvpeptide. An epitope can
comprise as few
as 3 amino acids in a spatial conformation which is unique to the epitope.
Generally an epitope
consists of at least 6 such amino acids, and more usually at least 8-10 such
amino acids.
Methods for determining the amino acids which make up an epitope include x-ray
crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping
e.g. the
Pepscan method described by Geysen et al. 1984: PCT Publication No. WO
84/03564; and PCT
Publication No. WO 84/03506.
Throughout the present specification, the expression "nucleotide sequence" may
be
employed to designate either a polynucleotide or a nucleic acid. More
precisely, the expression
"nucleotide sequence" encompasses the nucleic material itself and is thus not
restricted to the
sequence information (i.e. the succession of letters chosen among the four
base letters) that
biochemically characterizes a specific DNA or RNA molecule.
As used interchangeably herein, the terms "nucleic acids". "oligonucleotides",
and
"polynucleotides" include RNA, DNA, or RNA/DNA hybrid sequences of more than
one
nucleotide in either single chain or duplex form. The term "nucleotide" as
used herein as an
adjective to describe molecules comprising RNA. DNA, or RNA/DNA hybrid
sequences of any
length in single-stranded or duplex form. The term "nucleotide" is also used
herein as a noun to
refer to individual nucleotides or varieties of nucleotides, meaning a
molecule, or individual unit
in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose
or deoxyribose
sugar moiety, and a phosphate group, or phosphodiester linkage in the case of
nucleotides within
an oligonucleotide or polynucleotide. Although the term "nucleotide" is also
used herein to
encompass "modified nucleotides" which comprise at least one modifications (a)
an alternative
linking group, (b) an analogous form of purine, (c) an analogous form of
pyrimidine, or (d) an
analogous sugar, for examples of analogous linking groups, purine.
pyrimidines, and sugars see
for example PCT publication No. WO 95/04064. The polynucleotide sequences of
the invention
may be prepared by any known method, including synthetic, recombinant, ex vivo
generation, or
a combination thereof, as well as utilizing any purification methods known in
the art.
A sequence which is "operablv linked" to a regulatory sequence such as a
promoter
means that said regulatory element is in the correct location and orientation
in relation to the
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nucleic acid to control RNA polymerase initiation and expression of the
nucleic acid of interest.
As used herein, the term "operably linked" refers to a linkage of
polynucleotide elements in a
functional relationship. For instance, a promoter or enhancer is operably
linked to a coding
sequence if it affects the transcription of the coding sequence.
5 The terms "trait" and "phenotype" are used interchangeably herein and refer
to any
visible, detectable or otherwise measurable property of an organism such as
symptoms of, or
susceptibility to a disease for example. Typically the terms "trait" or
"phenotype" are used
herein to refer to symptoms of, or susceptibility to cancer or prostate
cancer, the level of
aggressiveness of cancer or prostate cancer tumors. an early onset of cancer
or prostate cancer, a
10 beneficial response to or side effects related to treatment against cancer
or prostate cancer.
The term "allele" is used herein to refer to variants of a nucleotide
sequence. A biallelic
polymorphism has two forms. Typically the first identified allele is
designated as the original
allele whereas other alleles are designated as alternative alleles. Diploid
organisms may be
homozygous or heterozygous for an allelic form.
The term "heterozygositv rate" is used herein to refer to the incidence of
individuals in a
population which are heterozygous at a particular allele. In a biallelic
system, the heterozygosity
rate is on average equal to 2Pa(1-Pa), where Pa is the frequency of the least
common allele. In
order to be useful in genetic studies, a genetic marker should have an
adequate level of
heterozygosity to allow a reasonable probability that a randomly selected
person will be
heterozygous.
The term " eno e" as used herein refers the identity of the alleles present in
an
individual or a sample. In the context of the present invention, a genotype
preferably refers to
the description of the biallelic marker alleles present in an individual or a
sample. The term
"genotyping" a sample or an individual for a biallelic marker consists of
determining the specific
allele or the specific nucleotide carried by an individual at a biallelic
marker.
The term "mutation" as used herein refers to a difference in DNA sequence
between or
among different genomes or individuals wiiich has a frequency below M.
The term "haplotvpe" refers to a combination of alleles present in an
individual or a
sample. In the context of the present invention. a haplotype preferably refers
to a combination of
biallelic marker alleles found in a given individual and which may be
associated with a
phenotype.
The term "polymorphism" as used herein refers to the occurrence of two or more
alternative genomic sequences or alleles between or among different genomes or
individuals.
"Polymorphic" refers to the condition in which two or more variants of a
specific genomic
sequence can be found in a population. A"polvmorphic site" is the locus at
which the variation
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occurs. A single nucleotide polymorphism is the replacement of one nucleotide
by another
nucleotide at the polymorphic site. Deletion of a singie nucleotide or
insertion of a single
nucleotide also gives rise to single nucleotide polymorphisms. In the context
of the present
invention, "single nucleotide polymorphism" preferably refers to a single
nucleotide substitution.
Typically, between different individuals, the polymorphic site may be occupied
by two different
nucleotides.
The term "biallelic polymorphism" and "biallelic marker" are used
interchangeably
herein to refer to a polymorphism, usually a single nucleotide, having two
alleles at a fairly high
frequency in the population. A"bialielic marker allele" refers to the
nucleotide variants present
at a biallelic marker site. Typically, the frequency of the less common allele
of the biallelic
markers of the present invention has been validated to be greater than 1%,
preferably the
frequency is greater than 10%, more preferably the frequency is at least 20%
(i.e. heterozygosity
rate of at least 0.32), even more preferably the frequency is at least 30%
(i.e. heterozygosity rate
of at least 0.42). A biallelic marker wherein the frequency of the less common
allele is 30% or
more is termed a "high quality biallelic marker".
As used herein the term "purH-related biallelic marker " relates to a set of
biallelic
markers in linkage disequilibrium with the purH gene. The term purH-related
biallelic marker
encompasses all of the biallelic markers A 1 to A43 disclosed in Table 2.
The location of nucleotides in a polynucleotide with respect to the center of
the
polynucleotide are described herein in the following manner. When a
polvnucleotide has an odd
number of nucleotides, the nucleotide at an equal distance from the 3' and 5'
ends of the
polynucleotide is considered to be "at the center" of the polynucleotide, and
any nucleotide
immediately adjacent to the nucleotide at the center, or the nucleotide at the
center itself is
considered to be "within I nucleotide of the center." With an odd number of
nucleotides in a
polynucleotide any of the five nucleotides positions in the middle of the
polvnucleotide would be
considered to be within 2 nucleotides of the center, and so on. When a
polvnucleotide has an
even number of nucleotides, there would be a bond and not a nucleotide at the
center of the
polynucleotide. Thus, eitlier of the two central nucleotides would be
considered to be "within I
nucleotide of the center" and any of the four nucleotides in the middle of the
polynucleotide
would be considered to be ''within 2 nucleotides of the center", and so on.
For polymorphisms
which involve the substitution, insertion or deletion of I or more
nucleotides. the polvmorphism,
allele or biallelic marker is ''at the center" of a polvnucleotide if the
difference between the
distance from the substituted, inserted, or deleted polynucleotides of the
polymorphism and the
3' end of the polynucleotide, and the distance froin the substituted.
inserted. or deleted
5 polynucleotides of the polymorphism and the 5' end of the polvnucleotide is
zero or one
3
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nucleotide. If this difference is 0 to 3, then the polymorphism is considered
to be "within I
nucleotide of the center." If the difference is 0 to 5, the polymorphism is
considered to be
"within 2 nucleotides of the center." If the difference is 0 to 7, the
polymorphism is considered
to be "within 3 nucleotides of the center," and so on.
The terms "complementary" or "complement thereof' are used herein to refer to
the
sequences of polynucleotides which is capable of forming Watson & Crick base
pairing with
another specified polynucleotide throughout the entirety of the complementary
region. For the
purpose of the present invention, a first polynucleotide is deemed to be
complementary to a
second polynucleotide when each base in the first polynucleotide is paired
with its
complementary base. Complementary bases are, generally, A and T (or A and U),
or C and G.
"Complement" is used herein as a synonym from "complementary polynucleotide",
"complementary nucleic acid" and "complementary nucleotide sequence". These
terms are
applied to pairs of polynucleotides based solely upon their sequences and not
any particular set
of conditions under which the two polynucleotides would actually bind.
The term "non- enic" is used herein to describe purH-related biallelic
markers, as well
as polynucleotides and primers which occur outside the nucleotide positions
shown in the human
purH genomic sequence of SEQ ID No 1. The non-genic biallelic marker of the
purH gene
could either be located in an intergenic region or in an other gene. The term
"genic" is used
herein to describe purH -related biallelic markers as well as polynucleotides
and primers which
do occur in the nucleotide positions shown in the human purH genomic sequence
of SEQ ID No
l.
Variants and Fragments
1- Polynucleotides
The invention also relates to variants and fragments of the polvnucleotides
described herein,
particularly of apurH gene containing one or more biallelic markers according
to the invention.
Variants of polynucleotides, as the term is used herein, are polynucleotides
that differ
from a reference polynucleotide. A variant of a polvnucleotide may be a
naturally occurring
variant such as a naturally occurring allelic variant, or it may be a variant
that is not known to
occur naturally. Such non-naturally occurring variants of the polynucleotide
may be made by
mutagenesis techniques, including those appfied to polynucleotides. cells or
organisms.
Generally, differences are limited so that the nucleotide sequences of the
reference and the
variant are closely similar overall and, in many regions, identical.
Variants of polynucleotides according to the invention include, without being
limited to,
nucleotide sequences which are at least 95% identical to a polvnucleotide
selected from the group
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13
consisting of the nucleotide sequences of SEQ ID No I or to any polynucleotide
fragment of at least
12, 15, 18, 20, 25, 30, 50, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500,
600 or 1000 consecutive
nucleotides of a polynucleotide selected from the group consisting of the
nucleotide sequences of
SEQ ID No 1, and preferably at least 99% identical, more particularly at least
99.5% identical, and
most preferably at least 99.8% identical to a polynucleotide selected from the
group consisting of
the nucleotide sequences of SEQ ID No I or to any polynucleotide fragment of
at least 12, 15, 18,
20, 25, 30, 50, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600 or 1000
consecutive nucleotides
of a polynucleotide selected from the group consisting of the nucleotide
sequences of SEQ ID No 1.
Nucleotide changes present in a variant polynucleotide may be silent, which
means that
they do not alter the amino acids encoded by the polynucleotide. However,
nucleotide changes
may also result in amino acid substitutions, additions, deletions, fusions and
truncations in the
polypeptide encoded by the reference sequence. The substitutions, deletions or
additions may
involve one or more nucleotides. The variants may be altered in coding or non-
coding regions
or both. Alterations in the coding regions may produce conservative or non-
conservative amino
acid substitutions, deletions or additions.
In the context of the present invention, particularly preferred embodiments
are those in
which the polynucleotides encode polypeptides which retain substantially the
same biological
function or activity as the mature purH protein, or those in which the
polynucleotides encode
polypeptides which maintain or increase a particular biological activity,
while reducing a second
biological activity
A polynucleotide fragment is a polynucleotide having a sequence that is
entirely the
same as part but not all of a given nucleotide sequence, preferably the
nucleotide sequence of
a purH gene, and variants thereof. The fragment can be a portion of an intron
of a purH gene. It
can also be a portion of the regulatory regions of purH, preferably of the
promoter sequence of
the pzrrH gene. Preferably, such fragments comprise at least one of the
biallelic markers Al to
A43 or the complements thereto.
Such fragments may be "free-standing", i.e. not part of or fused to other
polynucleotides,
or they may be comprised within a single larger polynucleotide of which thev
form a part or
region. Indeed, several of these fragments mav be present within a single
larger polynucleotide.
Optionally, such fragments may consist of, or consist essentially of a
contiguous span of
at least 8, 10, 12, 15, 18, 20. 25, 35, 40, 50, 70. 80. 100, 250. 500 or 1000
nucleotides in length.
2- Polypeptides
The invention also relates to variants, fra'~ments. analogs and derivatives of
the
polypeptides described herein, including mutated purH proteins.
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14
The variant may be 1) one in which one or more of the amino acid residues are
substituted with a conserved or non-conserved amino acid residue and such
substituted amino
acid residue may or may not be one encoded by the genetic code, or 2) one in
which one or more
of the amino acid residues includes a substituent group, or 3) one in which
the mutated purH is
fused with another compound, such as a compound to increase the half-life of
the polypeptide
(for example, polyethylene glycol), or 4) one in which the additional amino
acids are fused to the
mutated purH, such as a leader or secretory sequence or a sequence which is
employed for
purification of the mutated purH or a preprotein sequence. Such variants are
deemed to be within
the scope of those skilled in the art.
A polypeptide fragment is a polypeptide having a sequence that entirely is the
same as
part but not all of a given polypeptide sequence, preferably a polypeptide
encoded by a parrH
gene and variants thereof.
A specific embodiment of a modified purH peptide molecule of interest
according to the
present invention, includes, but is not limited to, a peptide molecule which
is resistant to
proteolysis, is a peptide in which the -CONH- peptide bond is modified and
replaced by a
(CH2NH) reduced bond, a (NHCO) retro inverso bond, a(CH2-O) methylene-oxy
bond, a
(CH2-S) thiomethylene bond, a (CH2CH2) carba bond, a (CO-CH2) cetomethylene
bond, a
(CHOH-CH2) hydroxyethylene bond), a (N-N) bound, a E-alcene bond or also a -
CH=CH-
bond. The invention also encompasses a human purH polypeptide or a fragment or
a variant
thereof in which at least one peptide bond has been modified as described
above.
Such fragments may be "free-standing", i.e. not part of or fused to other
polypeptides, or
they may be comprised within a single larger polypeptide of which they form a
part or region.
However, several fragments may be comprised within a single larger
polypeptide.
As representative examples of polypeptide fragments of the invention, there
may be
mentioned those which have at least 6 amino acids, preferably at least 8 to 10
amino acids, more
preferably at least 12, 15. 20, 25. 30, 40, 50, or 100 amino acids long. A
specific embodiment of
a purH fragment is a fragment containing at least one amino acid mutation in
the purH protein.
Identity Between Nucleic Acids Or Polypeptides
The terms "percentage of sequence identity" and "percentage homology" are used
interchangeably herein to refer to comparisons among polynucleotides and among
polypeptides,
and are determined bv comparing two optimally aligned sequences over a
comparison window,
wherein the portion of the polynucleotide or polypeptide sequence in the
comparison window
may comprise additions or deletions (i.e., gaps) as compared to the reference
sequence (which
does not comprise additions or deletions) for optimal alignment of the two
sequences. The
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percentage is calculated by determining the number of positions at which the
identical nucleic
acid base or amino acid residue occurs in both sequences to yield the number
of matched
positions, dividing the number of matched positions by the total number of
positions in the
window of comparison and multiplying the result by 100 to yield the percentage
of sequence
5 identity. Homology is evaluated using any of the variety of sequence
comparison algorithms
and programs known in the art. Such algorithms and programs include, but are
by no means
limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman,
1988; Altschul et al., 1990; Thompson et al., 1994: Higgins et al., 1996;
Altschul et al., 1993).
In a particularly preferred embodiment, protein and nucleic acid sequence
homologies are
10 evaluated using the Basic Local Alignment Search Tool ("BLAST") which is
well known in the
art (see, e.g., Karlin and Altschul, 1990; Altschul et al., 1990, 1993, 1997).
In particular, five
specific BLAST programs are used to perform the following task:
(1) BLASTP and BLAST3 compare an amino acid query sequence against a protein
sequence database;
15 (2) BLASTN compares a nucleotide query sequence against a nucleotide
sequence
database;
(3) BLASTX compares the six-frame conceptual translation products of a query
nucleotide sequence (both strands) against a protein sequence database;
(4) TBLASTN compares a query protein sequence against a nucleotide sequence
database translated in all six reading frames (both strands); and
(5) TBLASTX compares the six-frame translations of a nucleotide query sequence
against the six-frame translations of a nucleotide sequence database.
The BLAST programs identify homologous sequences by identifying similar
segments,
which are referred to herein as "high-scoring segment pairs," between a query
amino or nucleic
acid sequence and a test sequence which is preferably obtained from a protein
or nucleic acid
sequence database. High-scoring segment pairs are preferably identified (i.e.,
aligned) by means
of a scoring matrix, many of which are known in the art. Preferably, the
scoring matrix used is
the BLOSUM62 matrix (Gonnet et al.. 1992; Henikoff and Henikoff, 1993). Less
preferably,
the PAM or PAM250 matrices may also be used (see. e.g., Schwartz and Dayhoff,
eds., 1978).
The BLAST programs evaluate the statistical significance of all high-scoring
segment pairs
identified, and preferably selects those segments which satisfy a user-
specified threshold of
significance, sucli as a user-specified percent homology. Preferablv, the
statistical significance
of a high-scoring segment pair is evaluated usinQ the statistical significance
formula of Karlin
(see, e.g., Karlin and Altschul, 1990).
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16
Stringent Hybridization Conditions
For the purpose of defining such a hybridizing nucleic acid according to the
invention,
the stringent hybridization conditions are the followings :
the hybridization step is realized at 65 C in the presence of 6 x SSC buffer,
5 x
Denhardt's solution, 0,5% SDS and 100 g/ml of salmon sperm DNA.
The hybridization step is followed by four washing steps :
- two washings during 5 min, preferably at 65 C in a 2 x SSC and 0.1%SDS
buffer;
- one washing during 30 min, preferably at 65 C in a 2 x SSC and 0.1% SDS
buffer,
- one washing during 10 min, preferably at 65 C in a 0.1 x SSC and 0.1%SDS
buffer,
these hybridization conditions being suitable for a nucleic acid molecule of
about 20
nucleotides in length. There is no need to say that the hybridization
conditions described above
are to be adapted according to the length of the desired nucleic acid,
following techniques well
known to the one skilled in the art. The suitable hybridization conditions may
for example be
adapted according to the teachings disclosed in the book of Hames and Higgins
(1985).
Genomic Sequences Of The purH Gene
The present invention concerns the genomic sequence of purH. The present
invention
encompasses thepurH gene, or purH genomic sequences consisting of, consisting
essentially of,
or comprising the sequence of SEQ ID No 1, a sequence complementary thereto,
as well as
fragments and variants thereof. These polynucleotides may be purified,
isolated, or recombinant.
The invention also encompasses a purified, isolated, or recombinant
polynucleotide
comprising a nucleotide sequence having at least 70, 75, 80, 85. 90, or 95%
nucleotide identity
with a nucleotide sequence of SEQ ID No I or a complementary sequence thereto
or a fragment
thereof. The nucleotide differences as regards to the nucleotide sequence of
SEQ ID No 1 may
be generally randomly distributed throughout the entire nucleic acid.
Nevertheless, preferred
nucleic acids are those wherein the nucleotide differences as regards to the
nucleotide sequence
of SEQ ID No 1 are predominantly located outside the coding sequences
contained in the exons.
These nucleic acids, as well as their fragments and variants, may be used as
oligonucleotide
primers or probes in order to detect the presence of a copy of the pzrrH gene
in a test sample, or
alternatively in order to amplify a target nucleotide sequence within the purH
sequences.
Another object of the invention consists of a purified, isolated, or
recombinant nucleic
acid that hvbridizes with the nucleotide sequence of SEQ ID No I or a
complementarv sequence
thereto or a variant thereof, under the strinaent hvbridization conditions as
defined above.
Preferred nucleic acids of the invention include isolated, puritied, or
recombinant
polynucleotides comprising a contiguous span of at least 12, 15. 18. 20. 25.
30. 35, 40, 50, 60,
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17
70, 80, 90, 100, 150, 200, 400, 500, or 1000 nucleotides of SEQ ID No I or the
complements
thereof. Particularly preferred nucleic acids of the invention include
isolated, purified, or
recombinant polynucleotides comprising a contiguous span of at least 12, 15.
18, 20, 25, 30, 35,
40, 50, 60, 70, 80, 90, 100, 150, 200, 400, 500, or 1000 nucleotides of SEQ ID
No I or the
complements thereof, wherein said contiguous span comprises at least 1, 2, 3,
5, or 10 of the
following nucleotide positions of SEQ ID No 1: 1-1587, 1729-2000, 2095-2414,
2558-3235,
3848-3991, 4156-7043, 7396-7958, 8237-9596, 9666-9874, 9921-10039, 1 0083-1 1
742, 11825-
15173, 15267-15916, 16075-16750, 16916-22304, 22443-23269, 23384-24834, 24927-
25952,
26048-28683, 28829-34694, 37282-37458, 37765-37894, 38563-38932, 39178-39451,
39692-
39821, 40038-40445, and 40846-41587. Additional preferred nucleic acids of the
invention
include isolated, purified, or recombinant polynucleotides comprising a
contiguous span of at
least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90. 100, 150, 200, 400,
500, or 1000
nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous
span
comprises either a G at position 15234, or a G at position 36801 of SEQ ID No
1. Futher
preferred nucleic acids of the invention include isolated, purified, or
recombinant
polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25,
30, 35, 40, 50, 60,
70, 80, 90, 100, 150, 200, 400, 500, or 1000 nucleotides of SEQ ID No 1 or the
complements
thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of
the following
nucleotide positions of SEQ ID No 1: 1-1587, 1729-2000. 2095-2414, 2558-3235,
3848-3991,
4156-5000, 5001-6000, 6001-7043, 7396-7958, 8237-9596, 9666-9874, 9921-10039,
10083-
11742, 11825-13000, 13001-14000, 14001-15173, 15267-15916, 16075-16750, 16916-
18000,
18001-19000, 1 900 1-20000, 20001-21000, 21001-22304. 22443-23269, 23384-
24834, 24927-
25952, 26048-27000, 27001-28000, 28001-28683, 28829-30000, 30001-31000, 3 1001-
32000,
32001-33000, 33001-34694, 37282-37458, 37765-37894, 38563-38932, 39178-39451,
39692-
39821, 40038-40445, and 40846-4 1 5 87. It should be noted that nucleic acid
fragments of any
size and sequence may also be comprised by the polvnucleotides described in
this section.
ThepzrrHgenomic nucleic acid comprises 16 exons. The exon positions in SEQ ID
No
1 are detailed below in Table A.
Thus, the invention embodies purified, isolated, or recombinant
polvnucleotides
comprising a nucleotide sequence selected from tiie group consisting of the 16
exons of the purH
gene, or a sequence complementary thereto. The invention also deals with
purified, isolated, or
recombinant nucleic acids comprising a combination of at least two exons of
the pzn=H gene,
wherein the polvnucleotides are arranged within the nucleic acid. from the 5'-
end to the 3'-end
of said nucleic acid, in the same order as in SEQ ID No 1.
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Intron I refers to the nucleotide sequence located between Exon I and Exon 2,
and so
on. The position of the introns is detailed in Table A. Thus, the invention
embodies purified,
isolated, or recombinant polynucleotides comprising a nucleotide sequence
selected from the
group consisting of the 15 introns of the purH gene, or a sequence
complementary thereto.
Table A
Exon Position in SEQ ID No I Intron Position in SEQ ID No I
BeQinnin End Beginning End
1 2001 2096 1 2097 2432
2 2433 2559 2 2560 8091
3 8092 8168 3 8169 9599
4 9600 9666 4 9667 15177
5 15178 15266 5 15267 15923
6 15924 16075 6 16076 16758
7 16759 16915 7 16916 22308
8 22309 22434 8 22435 23276
9 23277 23384 9 23385 24840
24841 24926 10 24927 25956
11 25957 26046 11 26047 28699
12 28700 28828 12 28829 34698
13 34699 34791 13 34792 36678
14 36679 36861 14 36862 39013
39014 39169 15 39170 39455
16 39456 39684
The invention also concerns the polypeptide encoded by the nucieotide sequence
of SEQ
ID No 1, or a fragment thereof or a complementary sequence thereto.
While this section is entitled "Genomic Sequences of purH," it should be noted
that
10 nucleic acid fragments of any size and sequence may also be comprised by
the polvnucleotides
described in this section, flanking the genomic sequences of purHon either
side or between two
or more sucli genomic sequences.
purH cDNA Seguences
The expression of the pan-H gene lias been shown to lead to the production of
at least one
15 mRNA species, the nucleic acid sequence of which is set forth in SEQ ID No
2.
Another object of the invention is a purified. isolated, or recombinant
nucleic acid
comprising the nucleotide sequence of SEQ ID No 2. complementary sequences
thereto, as well
as allelic variants, and fragments thereof. Moreover. preferred
polynucleotides of the invention
include purified, isolated. or recombinant purHcDNAs consisting of, consisting
essentiallv of,
or comprising the sequence of SEQ ID No 2. Particularly preferred embodiments
of the
invention include isolated, purified, or recombinant polynucleotides
comprising a contiguous
span of at least 12, 15. 18. 20, 25. 30, 35. 40. 50. 60. 70, 80. 90, 100, 150,
200. 500. or 1000
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19
nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous
span
comprises a nucleotide selected in the group consisting of a G at position
424, and a G at
position 1520 of SEQ ID No 2.
The cDNA of SEQ ID No 2 includes a 5'-UTR region startina from the nucleotide
at
position 1 and ending at the nucleotide in position 77 of SEQ ID No 2. The
cDNA of SEQ ID
No 2 includes a 3'-UTR region starting from the nucleotide at position 1857
and ending at the
nucleotide at position 1965 of SEQ ID No 2. The polyadenylation site starts
from the nucleotide
at position 1938 and ends at the nucleotide in position 1943 of SEQ ID No 2.
Consequently, the invention concerns a purified, isolated, and recombinant
nucleic acid
comprising a nucleotide sequence of the 5'UTR of the pzrrH cDNA, a sequence
complementary
thereto, or an allelic variant thereof.
The invention also concerns the polypeptide encoded by the nucleotide sequence
of SEQ
ID No 2, or a fragment thereof or a complementary sequence thereto.
While this section is entitled "purHcDNA Sequences," it should be noted that
nucleic
acid fragments of any size and sequence may also be comprised by the
polynucleotides
described in this section, flanking the genomic sequences ofpurHon either side
or between two
or more such genomic sequences.
Reimlatorv Sequences Of purH
As mentioned, the genomic sequence of the purH gene contains regulatory
sequences
botli in the non-coding 5'-flanking region and in the non-coding 3'-flanking
region that border
the pzrrH coding region containing the three exons of this gene.
The 5'-regulatory sequence of the purH gene is localized between the
nucleotide in
position 1 and the nucleotide in position 2000 of the nucleotide sequence of
SEQ ID No 1. This
polynucleotide contains the promoter site. Three potential GC boxes are found
in the
5'regulatory sequence. They are located at 1833-1838, 1858-1863, and 1872-1877
of the
sequence of SEQ ID No 1. There is also a TATA box which is located at 1710-
1717 of the
sequence of SEQ ID No 1. The GC boxes and TATA box are known to be related to
a gene
promoter. Moreover, two others TATA box have been found in positions 727-734
(TATAAAAT) and 740-746 (TATAAAAT).
The 3'-regulatory sequence of the purH gene is localized between nucleotide
position
39685 and nucleotide position 41684 of SEQ ID No 1.
Polynucleotides derived from the 5' and 3' regulatory regions are useful in
order to
detect the presence of at least a copy of a nucleotide sequence of SEQ ID No I
or a fragment
thereof in a test sample.
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The promoter activity of the 5' regulatory regions contained in purH can be
assessed as
described below.
In order to identify the relevant biologicallv active polynucleotide fragments
or variants
of SEQ ID No 1, one of skill in the art will refer to the book of Sambrook et
al.(Sambrook,
5 1989) which describes the use of a recombinant vector carrying a marker gene
(i.e. beta
galactosidase, chloramphenicol acetyl transferase, etc.) the expression of
which will be detected
when placed under the control of a biologically active polynucleotide
fragments or variants of
SEQ ID No 1. Genomic sequences located upstream of the first exon of the purH
gene are
cloned into a suitable promoter reporter vector, such as the pSEAP-Basic,
pSEAP-Enhancer,
10 p(3gal-Basic, p(3ga1-Enhancer, or pEGFP-1 Promoter Reporter vectors
available from Clontech,
or pGL2-basic or pGL3-basic promoterless luciferase reporter gene vector from
Promega.
Briefly, each of these promoter reporter vectors include multiple cloning
sites positioned
upstream of a reporter gene encoding a readily assayable protein such as
secreted alkaline
phosphatase, luciferase, P galactosidase, or green fluorescent protein. The
sequences upstream
15 thepurHcoding region are inserted into the cloning sites upstream of the
reporter gene in both
orientations and introduced into an appropriate host cell. The level of
reporter protein is assayed
and compared to the level obtained from a vector which lacks an insert in the
cloning site. The
presence of an elevated expression level in the vector containing the insert
with respect to the
control vector indicates the presence of a promoter in the insert. If
necessary, the upstream
20 sequences can be cloned into vectors which contain an enhancer for
increasing transcription
levels from weak promoter sequences. A significant level of expression above
that observed
with the vector lacking an insert indicates that a promoter sequence is
present in the inserted
upstream sequence.
Promoter sequence within the upstream genomic DNA may be further defined by
constructing nested 5' and/or 3' deletions in the upstream DNA using
conventional techniques
such as Exonuclease III or appropriate restriction endonuclease digestion. The
resulting deletion
fragments can be inserted into the promoter reporter vector to determine
whether the deletion has
reduced or obliterated promoter activity, such as described, for example, by
Coles et al.(1998),
the disclosure of which is incorporated lierein by reference in its entirety.
In this way, the
boundaries of the promoters may be defined. If desired, potential individual
regulatory sites
within the promoter may be identified using site directed mutagenesis or
linker scanning to
obliterate potential transcription factor binding sites within the promoter
individually or in
combination. The effects of these mutations on transcription levels may be
determined by
inserting the mutations into cloning sites in promoter reporter vectors. This
type of assay is
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21
well-known to those skilled in the art and is described in WO 97/17359, US
Patent No.
5,374,544; EP 582 796; US Patent No. 5,698,389; US 5,643,746; US Patent No.
5,502,176; and
US Patent 5,266,488; the disclosures of which are incorporated by reference
herein in their
entirety.
The strength and the specificity of the promoter of the pzrrH gene can be
assessed
through the expression levels of a detectable polynucleotide operably linked
to the purH
promoter in different types of cells and tissues. The detectable
polynucleotide may be either a
polynucleotide that specifically hybridizes with a predefined oligonucleotide
probe, or a
polynucleotide encoding a detectable protein, including apurH polypeptide or a
fragment or a
variant thereof. This type of assay is well-known to those ski lled in the art
and is described in
US Patent No. 5,502,176; and US Patent No. 5,266.488; the disclosures of which
are
incorporated by reference herein in their entirety. Some of the methods are
discussed in more
detail below.
Polynucleotides carrying the regulatory elements located at the 5' end and at
the 3' end
of the pzrrHcoding region may be advantageously used to control the
transcriptional and
translational activity of an heterologous polynucleotide of interest.
Thus, the present invention also concerns a purified or isolated nucleic acid
comprising a
polynucleotide which is selected from the group consisting of the 5' and 3'
regulatory regions, or
a sequence complementary thereto or a biologically active fragment or variant
thereof.
The invention also pertains to a purified or isolated nucleic acid comprising
a
polynucleotide having at least 95% nucleotide identity with a polynucleotide
selected from the
group consisting of the 5' and 3' regulatory regions. advantageously 99 %
nucleotide identity,
preferably 99.5% nucleotide identity and most preferably 99.8% nucleotide
identity with a
polynucleotide selected from the group consistinQ of the 5' and 3' regulatory
regions, or a
sequence complementary thereto or a variant thereof or a biologically active
fragment thereof.
Another object of the invention consists of purified, isolated or recombinant
nucleic
acids comprising a polvnucleotide that livbridizes, under the stringent
hybridization conditions
defined herein, with a polynucleotide selected froin the group consisting of
the nucleotide
sequences of the 5'- and 3' re-ulatorv regions, or a sequence complementary
thereto or a variant
thereof or a biologically active fragment thereof.
Preferred fragments of the 5' regulatory region have a length of about 1500 or
1000
nucleotides, preferably of about 500 nucleotides, more preferably about 400
nucleotides, even
more preferably 300 nucleotides and most preferably about 200 nucleotides.
Preferred fragments of the 3' regulatory region are at least 50. 100, 150,
200, 300 or 400
5 bases in length.
3
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22
"Biologically active" polynucleotide derivatives of SEQ ID No I are
polynucleotides
comprising or alternatively consisting in a fragment of said polynucleotide
which is functional as
a regulatory region for expressing a recombinant polypeptide or a recombinant
polvnucleotide in
a recombinant cell host. It could act either as an enhancer or as a repressor.
For the purpose of the invention, a nucleic acid or polynucleotide is
"functional" as a
regulatory region for expressing a recombinant polypeptide or a recombinant
polynucleotide if
said regulatory polynucleotide contains nucleotide sequences which contain
transcriptional and
translational regulatory information, and such sequences are "operably linked"
to nucleotide
sequences which encode the desired polypeptide or the desired polynucleotide.
The regulatory polynucleotides of the invention may be prepared from the
nucleotide
sequence of SEQ ID No I by cleavage using suitable restriction enzvmes, as
described for
example in the book of Sambrook et al.(1989). The regulatory polynucleotides
may also be
prepared by digestion of SEQ ID No I by an exonuclease enzyme, such as Bal31
(Wabiko et al.,
1986). These regulatory polynucleotides can also be prepared by nucleic acid
chemical
synthesis, as described elsewhere in the specification.
The regulatory polynucleotides according to the invention may be part of a
recombinant
expression vector that may be used to express a coding sequence in a desired
host cell or host
organism. The recombinant expression vectors according to the invention are
described
elsewhere in the specification.
A preferred 5'-regulatory polynucleotide of the invention includes the 5'-UTR
of the
pzrrHcDNA, or a biologically active fragment or variant thereof.
A preferred 3'-regulatory polynucleotide of the invention includes the 3'-UTR
of the
purHcDNA, or a biologically active fragment or variant thereof.
A further object of the invention consists of a purified or isolated nucleic
acid
comprising:
a) a nucleic acid comprising a regulatory nucleotide sequence selected from
the group
consisting of:
(i) a nucleotide sequence comprising a polvnucleotide of the 5' regulatory
region or a
complementary sequence thereto;
(ii) a nucleotide sequence comprising a polvnucleotide having at least 95% of
nucleotide
identity with the nucleotide sequence of the 5' regulatory region or a
complementary sequence
thereto;
(iii) a nucleotide sequence comprising a polvnucleotide that hvbridizes under
stringent
liybridization conditions with the nucleotide sequence of tiie 5' regulatory
region or a
3
5 complementary sequence thereto; and
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23
(iv) a biologically active fragment or variant of the polynucleotides in (i),
(ii) and (iii);
b) a polynucleotide encoding a desired polypeptide or a nucleic acid of
interest, operably
linked to the nucleic acid defined in (a) above;
c) Optionally, a nucleic acid comprising a 3'- regulatory polynucleotide,
preferably a 3'-
regulatory polynucleotide of the purH gene.
The regulatory polynucleotide of the 5' regulatory region, or its biologically
active
fragments or variants, is operably linked at the 5'-end of the polynucleotide
encoding the desired
polypeptide or polynucleotide.
The regulatory polynucleotide of the 3' regulatory region, or its biologically
active
fragments or variants, is advantageously operably linked at the 3'-end of the
polynucleotide
encoding the desired polypeptide or polynucleotide.
The desired polypeptide encoded by the above-described nucleic acid may be of
various
nature or origin, encompassing proteins of prokarvotic or eukarvotic origin.
Among the
polypeptides expressed under the control of a pzrrH regulatory region include
bacterial, fungal or
viral antigens. Also encompassed are eukaryotic proteins such as intracellular
proteins, like
"house keeping" proteins, membrane-bound proteins, like receptors, and
secreted proteins like
endogenous mediators such as cytokines. The desired polypeptide may be the
purH protein,
especially the protein of the amino acid sequence of SEQ ID No 3, or a
fragment or a variant
thereof.
The desired nucleic acids encoded by the above-described polvnucleotide,
usually an
RNA molecule, may be complementary to a desired coding polynucleotide, for
example to the
purH coding sequence, and thus useful as an antisense polynucleotide.
Such a polynucleotide may be included in a recombinant expression vector in
order to
express the desired polypeptide or the desired nucleic acid in host cell or in
a host organism.
Suitable recombinant vectors that contain a polynucleotide such as described
herein are
disclosed elsewhere in the specification.
CodinE Reljons
The pzrrHopen reading frame is contained in the corresponding mRNA of SEQ ID
No 2.
More precisely, the effective pzrrHcoding sequence (CDS) includes the region
between
nucleotide position 78 (first nucleotide of the ATG codon) and nucleotide
position 1856 (end
nucleotide of the TGA codon) of SEQ ID No 2. The present invention also
embodies isolated,
purified, and recombinant polvnucleotides which encode a polypeptides
comprising a contiguous
span of at least 6 amino acids, preferably at least 8 or 10 amino acids. more
preferably at least
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24
12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 3, wherein said
contiguous span
includes a serine residue at amino acid position 116 in SEQ ID No 3.
The above disclosed polynucleotide that contains the coding sequence of the
purH gene
may be expressed in a desired host cell or a desired host organism, when this
polynucleotide is
placed under the control of suitable expression signals. The expression
signals may be either the
expression signals contained in the regulatory regions in the purH gene of the
invention or in
contrast the signals may be exogenous regulatory nucleic sequences. Such a
polynucleotide,
when placed under the suitable expression signals, may also be inserted in a
vector for its
expression and/or amplification.
Polynucleotide Constructs
The terms "polynucleotide construct" and ''recombinant polynucleotide" are
used
interchangeably herein to refer to linear or circular, purified or isolated
polvnucleotides that have
been artificially designed and which comprise at least two nucleotide
sequences that are not
found as contiguous nucleotide sequences in their initial natural environment.
DNA Construct That Enables Directing Temporal And Spatial purH Gene
Expression In Recombinant Cell Hosts And In Transgenic Animals.
In order to study the physiological and phenotypic consequences of a lack of
synthesis of
the purH protein, both at the cell level and at the multi cellular organism
level, the invention also
encompasses DNA constructs and recombinant vectors enabling a conditional
expression of a
specific allele of the purH genomic sequence or cDNA and also of a copy of
this genomic
sequence or cDNA harboring substitutions, deletions, or additions of one or
more bases as
regards to the purH nucleotide sequence of SEQ ID Nos 1 and 2, or a fragment
thereof, these
base substitutions, deletions or additions being located either in an exon, an
intron or a
regulatory sequence, but preferably in the 5'-regulatory sequence or in an
exon of the purH
genomic sequence or within the pzrrHcDNA of SEQ ID No 2. In a preferred
embodiment, the
purH sequence comprises a biallelic marker of the present invention. ln a
preferred
embodiment, the pzirH sequence comprises a biallelic marker of the present
invention, preferably
one of the biallelic markers A l to A17, A34 and A35.
The present invention embodies recombinant vectors comprising anv one of the
polynucleotides described in the present invention. More particularly, the
polvnucleotide
constructs according to the present invention can comprise any of the
polvnucleotides described
in the "Genomic Sequences Of The HLunan purH Gene" section, the " purH cDNA
Sequences"
section, the "Coding Regions" section. and the "Oligonucleotide Probes And
Primers" section.
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A first preferred DNA construct is based on the tetracycline resistance operon
tet from
E. coli transposon Tn 10 for controlling the pzrrH gene expression, such as
described by Gossen
et al.(1992, 1995) and Furth et al.(1994). Such a DNA construct contains seven
tet operator
sequences from Tn 10 (tetop) that are fused to either a minimal promoter or a
5'-regulatory
5 sequence of the purH gene, said minimal promoter or said purH regulatory
sequence being
operably linked to a polynucleotide of interest that codes either for a sense
or an antisense
oligonucleotide or for a polypeptide, including a pztrH polypeptide or a
peptide fragment thereof.
This DNA construct is functional as a conditional expression system for the
nucleotide sequence
of interest when the same cell also comprises a nucleotide sequence coding for
either the wild
10 type (tTA) or the mutant (rTA) repressor fused to the activating domain of
viral protein VP16 of
herpes simplex virus, placed under the control of a promoter, such as the
HCMVIE I
enhancer/promoter or the MMTV-LTR. Indeed. a preferred DNA construct of the
invention
comprise both the polynucleotide containing the tet operator sequences and the
polynucleotide
containing a sequence coding for the tTA or the rTA repressor.
15 In a specific embodiment, the conditional expression DNA construct contains
the
sequence encoding the mutant tetracycline repressor rTA, the expression of the
polynucleotide
of interest is silent in the absence of tetracycline and induced in its
presence.
DNA Constructs Allowing Homologous Recombination: Replacement Vectors
A second preferred DNA construct will comprise, from 5'-end to 3'-end: (a) a
first
20 nucleotide sequence that is comprised in the pzrrH genomic sequence; (b) a
nucleotide sequence
comprising a positive selection marker, such as the marker for neomycine
resistance (neo); and
(c) a second nucleotide sequence that is comprised in the purH genomic
sequence, and is located
on the genome downstream the first purH nucleotide sequence (a).
In a preferred embodiment, this DNA construct also comprises a negative
selection
25 marker located upstream the nucleotide sequence (a) or downstream the
nucleotide sequence (c).
Preferably, the negative selection marker consists of the thymidine kinase
(tk) gene (Thomas et
al., 1986), the hygromycine beta gene (Te Riele et al., 1990), the hprt gene (
Van der Lugt et al.,
1991; Reid et al., 1990) or the Diphteria toxin A fragment (Dt-A) gene (Nada
et al., 1993; Yagi
et al.1990). Preferably, the positive selection marker is located within a
pzrrH exon sequence so
as to interrupt the sequence encodin(y a purH protein. These replacement
vectors are described,
for example, by Thomas et al.(1986: 1987), ivlansour et al.(1988) and Koller
et al.(1992).
The first and second nucleotide sequences (a) and (c) may be indifferently
located
within a purH regulatory sequence, an intronic sequence. an exon sequence or a
sequence
containina both regulatorv and/or intronic andior exon sequences. The size of
the nucleotide
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26
sequences (a) and (c) ranges from I to 50 kb, preferably from I to 10 kb, more
preferably from 2
to 6 kb and most preferably from 2 to 4 kb.
DNA Constructs Allowing Homologous Recombination: Cre-LoxP System.
These new DNA constructs make use of the site specific recombination system of
the P1
phage. The P1 phage possesses a recombinase called Cre which interacts
specifically with a 34
base pairs loxP site. The loxP site is composed of two palindromic sequences
of 13 bp separated
by a 8 bp conserved sequence (Hoess et al., 1986). The recombination by the
Cre enzyme
between two loxP sites having an identical orientation leads to the deletion
of the DNA
fragment.
The Cre-loxP system used in combination with a homologous recombination
technique
has been first described by Gu et al.(1993, 1994). Briefly, a nucleotide
sequence of interest to be
inserted in a targeted location of the genome harbors at least two IoxP sites
in the same
orientation and located at the respective ends of a nucleotide sequence to be
excised from the
recombinant genome. The excision event requires the presence of the
recombinase (Cre)
enzyme within the nucleus of the recombinant cell liost. The recombinase
enzyme may be
brought at the desired time either by (a) incubating the recombinant cell
hosts in a culture
medium containing this enzyme, by injecting the Cre enzyme directly into the
desired cell, such
as described by Araki et al.(1995), or by lipofection of the enzyme into the
cells, such as
described by Baubonis et al.(1993); (b) transfecting the cell host with a
vector comprising the
Cre coding sequence operably linked to a promoter functional in the
recombinant cell host,
which promoter being optionally inducible, said vector being introduced in the
recombinant cell
host, such as described by Gu et al.(1993) and Sauer et al.(1988); (c)
introducing in the genome
of the cell host a polynucleotide comprising the Cre coding sequence operably
linked to a
promoter functional in the recombinant cell liost, which promoter is
optionallv inducible, and
said polynucleotide being iiiserted in tfie genome of the cell host either by
a random insertion
event or an homologous recombination event, such as described by Gu et al.(
1994).
In a specific embodiment, the vector containing the sequence to be inserted in
the pz+rH
gene by homologous recombination is constructed in such a way that selectable
markers are
flanked by loxP sites of the same orientation, it is possible, by treatment by
the Cre enzvme, to
eliminate the selectable markers while leaving the purH sequences of interest
that have been
inserted by an homologous recombination event. Auain, two selectable markers
are needed: a
positive selection marker to select for the recombination event and a negative
selection marker to
select for the homologous recombination event. Vectors and methods using the
Cre-loxP system
are described by Zou et al.(1994).
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Thus, a third preferred DNA construct of the invention comprises, from 5'-end
to 3'-
end: (a) a first nucleotide sequence that is comprised in the purH genomic
sequence; (b) a
nucleotide sequence comprising a polvnucleotide encoding a positive selection
marker, said
nucleotide sequence comprising additionally two sequences defining a site
recognized by a
recombinase, such as a loxP site, the two sites being placed in the same
orientation; and (c) a
second nucleotide sequence that is comprised in the purH genomic sequence, and
is located on
the genome downstream of the first purH nucleotide sequence (a).
The sequences defining a site recognized by a recombinase, such as a loxP
site, are
preferably located within the nucleotide sequence (b) at suitable locations
bordering the
nucleotide sequence for which the conditional excision is sought. In one
specific embodiment,
two loxP sites are located at each side of the positive selection marker
sequence, in order to
allow its excision at a desired time after the occurrence of the homologous
recombination event.
In a preferred embodiment of a method using the third DNA construct described
above,
the excision of the polvnucleotide fragment bordered by the two sites
recognized by a
recombinase, preferably two loxP sites, is performed at a desired time, due to
the presence
within the genome of the recombinant host cell of a sequence encoding the Cre
enzyme operably
linked to a promoter sequence, preferably an inducible promoter, more
preferably a tissue-
specific promoter sequence and most preferably a promoter sequence which is
both inducible
and tissue-specific, such as described by Gu et al.(1994).
The presence of the Cre enzyme within the genome of the recombinant cell host
may
result of the breeding of two transgenic animals, the first transgenic animal
bearing the purH-
derived sequence of interest containing the loxP sites as described above and
the second
transgenic animal bearing the Cre coding sequence operably linked to a
suitable promoter
sequence, such as described by Gu et al.(1994).
Spatio-temporal control of the Cre enzyme expression may also be achieved with
an
adenovirus based vector that contains the Cre gene thus allowing infection of
cells, or in vivo
infection of organs, for delivery of the Cre enzyme. such as described by
Anton and Graham
(1995) and Kanegae et al.(1995).
The DNA constructs described above may be used to introduce a desired
nucleotide
sequence of the invention, preferablv a purH ;enomic sequence or a purH cDNA
sequence, and
most preferably an altered copy of a purH genomic or cDNA sequence, within a
predetermined
location of the targeted geilome. leading either to the generation of an
altered copy of a targeted
gene (knock-out homologous recombination) or to the replacement of a copy of
the targeted
gene by another copy sufficiently homologous to allow an homologous
recombination event to
occur (knock-in homologous recombination). In a specific embodiment. the DNA
constructs
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28
described above may be used to introduce a purH genomic sequence or a purHcDNA
sequence
comprising at least one biallelic marker of the present inveiition, preferably
at least one biallelic
marker selected from the group consisting of A I to A 17, A34 and A35.
Nuclear Antisense DNA Constructs
Other compositions containing a vector of the invention comprising an
oligonucleotide
fragment of the nucleic sequence SEQ ID No 2, preferably a fragment including
the start codon
of the purH geiie, as an antisense tool that inhibits the expression of the
corresponding purH
gene. Preferred methods using antisense polynucleotide according to the
present invention are
the procedures described by Sczakiel et al.(1995) or those described in PCT
Application No WO
95/24223, the disclosures of which are incorporated by reference herein in
their entirety.
Preferably, the antisense tools are chosen among the polynucleotides (15-200
bp long)
that are complementary to the 5'end of the purH mRNA. In one embodiment, a
combination of
different antisense polynucleotides complementary to different parts of the
desired targeted gene
are used.
Preferred antisense polynucleotides according to the present invention are
complementary to a sequence of the mRNAs of purH that contains either the
translation
initiation codon ATG or a splicing site. Further preferred antisense
polynucleotides according to
the invention are complementary of the splicing site of the purH mRNA.
Preferably, the antisense polynucleotides of the invention have a 3'
polyadenylation
signal that has been replaced with a self-cleaving ribozyme sequence, such
that RNA polymerase
II transcripts are produced without poly(A) at their 3' ends, these antisense
polynucleotides
being incapable of export from the nucleus, such as described by Liu et
al.(1994). In a preferred
embodiment, these purH antisense polynucleotides also comprise, within the
ribozyme cassette,
a histone stem-loop structure to stabilize cleaved transcripts against 3'-5'
exonucleolytic
degradation, such as the structure described by Eckner et al.(1991).
Oligonucleotide Probes And Primers
Polvnucleotides derived from the purH gene are useful in order to detect the
presence of
at least a copy of a nucleotide sequence of SEQ ID No 1, or a fragment,
complement, or variant
thereof in a test sample.
Particularly preferred probes and primers of the invention include isolated,
purified, or
recombinant polynucleotides comprising a contiguous span of at least 12, 15,
18. 20, 25, 30, 35,
40, 50. 60, 70, 80, 90, 100, 150, 200. 500, or 1000 nucleotides of SEQ ID No I
or the
complements thereof, wherein said contiguous span comprises at least 1, 2, 3 ,
5, or 10 of the
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following nucleotide positions of SEQ ID No 1: 1-1587, 1729-2000, 2095-2414,
2558-3235,
3848-3991, 4156-7043, 7396-7958, 8237-9596, 9666-9874, 9921-10039, 10083-
11742, 11825-
15173, 15267-15916, 16075-16750, 16916-22304, 22443-23269, 23384-24834, 24927-
25952,
26048-28683, 28829-34694, 37282-37458, 37765-37894, 38563-38932, 39178-39451,
39692-
39821, 40038-40445, and 40846-41587. Additional preferred probes and primers
of the
invention include isolated, purified, or recombinant polynucleotides
comprising a contiguous
span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,
200, 500, or 1000
nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous
span
comprises either a G at position 15234, or a G at position 36801 of SEQ ID No
1. Further
preferred probes and primers of the invention include isolated, purified, or
recombinant
polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25,
30, 35, 40, 50, 60,
70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No I or the
complements thereof,
wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the
following nucleotide
positions of SEQ ID No 1: 1-1587, 1729-2000, 2095-2414, 2558-3235, 3848-3991.
4156-5000,,
5001-6000, 6001-7043, 7396-7958, 8237-9596, 9666-9874, 9921-10039, 10083-
11742, 11825-
13000, 13001-14000, 14001-15173, 15267-15916. 16075-16750, 16916-18000, 18001-
19000,
19001-20000, 20001-21000, 21001-22304, 22443-23269, 23384-24834, 24927-25952,
26048-
27000, 27001-28000, 28001-28683, 28829-30000. 30001-31000, 3 1 00 1-32000,
32001-33000,
33001-34694, 37282-37458, 37765-37894, 38563-38932, 39 1 78-3945 1, 39692-
39821, 40038-
40445, and 40846-41587.
Another object of the invention is a purified, isolated, or recombinant
polynucleotide
comprising the nucleotide sequence of SEQ ID No 2, complementary sequences
thereto, as well
as allelic variants, and fragments thereof. Moreover, preferred primers and
probes of the
invention include purified, isolated, or recombinant purHcDNAs consisting of,
consisting
essentially of, or comprising the sequence of SEQ ID No 2. Particularly
preferred probes and
primers of the invention comprise a contiguous span of at least 12. 15, 18,
20, 25, 30. 35, 40, 50,
60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the
complements
thereof, wherein said contiguous span comprises a nucleotide selected in the
group consisting of
a G at position 424, and a G at position 1520 of SEQ ID No 2.
A further embodiment of the invention includes isolated, purified, or
recombinant
polynucleotides comprising a contiguous span of at least 12, 15, 18, 20. 25,
30, 35. 40, 50, 60,
70, 80, 90, 100, 150, 200, or 500 nucleotides. to the extent that such lengths
are consistent witli
the specific sequence, of a sequence selected from the group consisting of SEQ
ID Nos. 4 to 22,
and the complements thereof, optionally wherein said contiguous span comprises
either allele I
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or allele 2 of apurH-related biallelic marker selected from the group
consisting of A18 to A33
and A36 to A43.
Thus, the invention also relates to nucleic acid probes characterized in that
they
hybridize specifically, under the stringent hybridization conditions defined
above, with a nucleic
5 acid selected from the group consisting of the nucleotide sequences 1-1587,
1729-2000, 2095-
2414, 2558-3235, 3848-3991, 4156-7043, 7396-7958, 8237-9596, 9666-9874, 9921-
10039,
1 0083-1 1 742, 1 1 825-1 5 1 73, 15267-15916, 16075-16750, 1 69 1 6-223 04,
22443-23269, 23384-
24834, 24927-25952, 26048-28683, 28829-34694, 37282-37458, 37765-37894, 38563-
38932,
39178-39451, 39692-39821, 40038-40445, and 40846-4 1 5 87 of SEQ ID No I or a
variant
10 thereof or a sequence complementary thereto.
The formation of stable hybrids depends on the melting temperature (Tm) of the
DNA.
The Tm depends on the length of the primer or probe, the ionic strength of the
solution and the
G+C content. The higher the G+C content of the primer or probe, the higher is
the melting
temperature because G:C pairs are held by three H bonds whereas A:T pairs have
only two. The
15 GC content in the probes of the invention usually ranges between 10 and 75
%, preferably
between 35 and 60 %, and more preferably between 40 and 55 %.
A probe or a primer according to the invention has between 8 and 1000
nucleotides in
length, or is specified to be at least 12, 15, 18, 20, 25, 35, 40, 50. 60, 70,
80, 100, 250, 500 or
1000 nucleotides in length. More particularly, the length of these probes can
range from 8, 10,
20 15, 20, or 30 to 100 nucleotides, preferably from 10 to 50, more preferably
from 15 to 30
nucleotides. Shorter probes tend to lack specificity for a target nucleic acid
sequence and
generally require cooler temperatures to form sufficiently stable hvbrid
complexes with the
template. Longer probes are expensive to produce and can sometimes self-
hybridize to form
hairpin structures. The appropriate length for primers and probes under a
particular set of assay
25 conditions may be empirically determined by one of skill in the art.
A preferred probe or primer consists of a nucleic acid comprising a
polynucleotide
selected from the group of the nucleotide sequences of P1 to P42 and the
complementary
sequence thereto, B 1 to B34, C I to 04. D I to D42. E 1 to E42, for wh ich
the respective
locations in the sequence listing are provided in Tables 1, 2 and 3.
30 Additionally, another preferred embodiment of a probe according to the
invention
consists of a nucleic acid comprising a biallelic niarker selected from the
group consisting of A1
to A43 or the complements thereto, for which the respective locations in the
sequence listing are
provided in Table 2.
The invention also relates to a purified and/or isolated nucleotide sequence
comprising a
3
5 polymorphic base of a pau=H-related biallelic marker, preferably of a
biallelic marker selected
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from the group consisting of Al to A43, and the complements thereof. The
sequence has
between 8 and 1000 nucleotides in length, and preferably comprises at least 8,
10, 12, 15, 18, 20,
25, 35, 40, 50, 60, 70, 80, 100, 250, 500 or 1000 contiguous nucleotides, to
the extent that such
lengths are consistent with the specific sequence, of a nucleotide sequence
selected from the
group consisting of SEQ ID Nos 1, 2, and 4 to 22 or a variant thereof or a
complementary
sequence thereto. In one embodiment the invention encompasses isolated,
purified, and
recombinant polynucleotides consisting of, or consisting essentially of a
contiguous span of 8 to
50 nucleotides of any one of SEQ ID Nos 1, 2, or 4 to 22 and the complement
thereof, wherein
said span includes apurH-related biallelic marker in said sequence;
optionally, wherein said
purH-related biallelic marker is selected from the group consisting of A I to
A43, and the
complements thereof, or optionally the biallelic markers in linkage
disequilibrium therewith;
optionally, wherein said purH-related biallelic marker is selected from the
group consisting of
Al, A3 to A 14, A16 to A 17, A34, and A35, and the complements thereof, or
optionally the
biallelic markers in linkage disequilibrium therewith; optionally, wherein
said purH-related
biallelic marker is selected from the group consisting of A2 and A 15, and the
complements
thereof, or optionally the biallelic markers in linkage disequilibrium
therewith; optionally,
wherein said purH-related biallelic marker is selected from the group
consisting of Al8 to A33
and A36 to A43; optionally, wherein said purH-related biallelic marker is
selected from the
group consisting of A29, A7, A20, A 10 and A 13, and the complements thereof,
or optionally the
biallelic markers in linkage disequilibrium therewith: optionally, wherein
said purH-related
biallelic marker is selected from the group consisting of A30, A17. A28, A25,
A21, and A14,
and the complements thereof, or optionally the biallelic markers in linkage
disequilibrium
therewith. These nucleotide sequences comprise the polymorphic base of either
allele I or allele
2 of the considered biallelic marker. Optionally, said biallelic marker may be
within 6, 5, 4, 3, 2,
or I nucleotides of the center of said polynucleotide or at the center of said
polynucleotide;
optionally, wherein said contiguous span is 18 to 35 nucleotides in length and
said biallelic
marker is within 4 nucleotides of the center of said polynucleotide;
optionally, wherein said
polynucleotide consists of said contiguous span and said contiguous span is 25
nucleotides in
length and said biallelic marker is at the center of said polynucleotide;
optionally, wherein the 3'
end of said contiguous span is present at the 3' end of said polvnucleotide;
and optionally,
wherein the 3' end of said contiguous span is located at the 3' end of said
polvnucleotide and said
biallelic marker is present at the 3' end of said polynucleotide. Optionally,
said polvnucleotide
may fiirther comprise a label. Optionally, said polvnucleotide can be attached
to solid support.
In a further embodiment, the polynucleotides defined above can be used alone
or in any
combination. In a preferred embodiment, said probes comprises. consists of, or
consists
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32
essentially of a sequence selected from the following sequences: P 1 to P42
and the
complementary sequences thereto.
In another embodiment the invention encompasses isolated, purified and
recombinant
polynucleotides comprising, consisting of, or consisting essentially of a
contiguous span of 8 to
50 nucleotides of SEQ ID Nos. 1, 2, or 4 to 22 or the complements thereof,
wherein the 3' end of
said contiguous span is located at the 3' end of said polynucleotide, and
wherein the 3' end of
said polynucleotide is located within or at least 2, 4, 6, 8, 10, 12, 15, 18,
20, 25, 50, 100, 250,
500, or 1000 nucleotides upstream of apurH-related biallelic marker in said
sequence,
preferably within 20 nucleotides upstream; optionally, wherein said purH-
related biallelic
marker is selected from the group consisting of A I to A43, and the
complements thereof, or
optionally the biallelic markers in linkage disequilibrium therewith;
optionally, wherein said
purH-related biallelic marker is selected from the group consisting of Al, A3
to A14, A16 to
A17, A34, and A35, and the complements thereof, or optionally the biallelic
markers in linkage
disequilibrium therewith; optionally, wherein said purH-related biallelic
marker is selected from
the group consisting of A2 and A 15, and the complements thereof, or
optionally the biallelic
markers in linkage disequilibrium therewith; optionally, wherein said purH-
related biallelic
marker is selected from the group consisting of A 18 to A33 and A36 to A43;
optionally, wherein
said purH-related biallelic marker is selected from the group consisting of
A29, A7, A20, A10
and A13, and the complements thereof, or optionally the biallelic markers in
linkage
disequilibrium therewith; optionally, wherein said purH-related biallelic
marker is selected from
the group consisting of A30, A17, A28, A25, A21, and A14, and the complements
thereof, or
optionally the biallelic markers in linkage disequilibrium therewith;
optionally, wherein the 3'
end of said polynucleotide is located I nucleotide upstream of said purH-
related biallelic marker
in said sequence; and optionally, wherein said polvnucleotide consists
essentially of a sequence
selected from the following sequences: D I to D42 and E 1 to E42. Optionally,
said
polynucleotide may further comprise a label. Optionally, said polynucleotide
can be attached to
solid support. In a further embodiment, the polvnucleotides defined above can
be used alone or
in any combination.
In a further embodiment. the invention encompasses isolated, purified, or
recombinant
polynucleotides comprising, consisting of, or consisting essentially of a
sequence selected from
the followin- sequences: B 1 to B34 and C 1 to C34.
In an additional embodiment, the inveiition encompasses the use of any
polynucleotide
for, or polynucleotides for use in determining the identity of the nucleotide
at a purH-related
biallelic marker or the complements thereof, as well as polvnucleotides for
use or use of
polynucleotides in amplifving segments of nucleotides comprising apzu-H-
related biallelic
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
marker or the complements thereof: Optionally, said determining may be
performed in a
hybridization assay, sequencing assay, microsequencing assay, or an enzyme-
based mismatch
detection assay; Optionally, said amplifying may be performed by a PCR or LCR.
optionally,
wherein said purH-related biallelic marker is selected from the group
consisting of A1 to A43,
and the complements thereof, or optionally the biallelic markers in linkage
disequilibrium
therewith; optionally, wherein said purH-related biallelic marker is selected
from the group
consisting of A l, A3 to A 14, A l 6 to A 17, A34, and A3 5, and the
complements thereof, or
optionally the biallelic markers in linkage disequilibrium therewith;
optionally, wherein said
purH-related biallelic marker is selected from the group consisting of A2 and
A15, and the
complements thereof, or optionally the biallelic markers in linkage
disequilibrium therewith;
optionally, wherein said purH-related biallelic marker is selected from the
group consisting of
A18 to A33 and A36 to A43; optionally, wherein said purH-related biallelic
marker is selected
from the group consisting of A29, A7, A20, A 10 and A 13, and the complements
thereof, or
optionally the biallelic markers in linkage disequilibrium therewith;
optionally, wherein said
purH-related biallelic marker is selected from the group consisting of A30,
A17, A28, A25, A21,
and A14, and the complements thereof, or optionally the biallelic markers in
linkage
disequilibrium therewith; Optionally, said polynucleotide may be attached to a
solid support,
array, or addressable array; Optionally, said polvnucleotide may be labeled.
The invention concerns the use of the polynucleotides according to the
invention for
determining the identity of the nucleotide at apurH-related biallelic marker,
preferably in
hybridization assay, sequencing assay, microsequencing assay, or an enzyme-
based mismatch
detection assay and in amplifying segments of nucleotides comprising aparrH-
related biallelic
marker. In addition, the polvnucleotides of the invention for use or the use
of polynucleotides in
determining the identity of one or more nucleotides at apurH-related biallelic
marker encompass
polynucleotides with any further limitation described in this disclosure, or
those following,
specified alone or in any combination.
The primers and probes can be prepared by any suitable method, including, for
example,
cloning and restriction of appropriate sequences and direct chemical synthesis
by a method such
as the phosphodiester method of Narang et al.(1979), the phosphodiester method
of Brown et
al.(1979), the diethylphosphoramidite method of Beaucage et al.(l 981) and the
solid support
method described in EP 0 707 592. The disclosures of the preceding documents
are incorporated
herein bv reference in their entirety.
Detection probes are generally nucleic acid sequences or uncharged nucleic
acid analogs
such as, for example peptide nucleic acids which are disclosed in
International Patent
Application WO 92/20702. morpholino analogs which are described in U.S.
Patents Numbered
CA 02368672 2001-09-24
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34
5,185,444; 5,034,506 and 5,142,047. The probe may have to be rendered "non-
extendable" in
that additional dNTPs cannot be added to the probe. In and of themselves
analogs usually are
non-extendable and nucleic acid probes can be rendered non-extendable by
modifying the 3' end
of the probe such that the hydroxyl group is no longer capable of
participating in elongation. For
example, the 3' end of the probe can be functionalized with the capture or
detection label to
thereby consume or otherwise block the hvdroxyl group. Alternatively, the 3'
hvdroxyl group
simply can be cleaved, replaced or modified. U.S. Patent Application Serial
No. 07/049,061
filed April 19, 1993 describes modifications, which can be used to render a
probe non-
extendable.
Any of the polynucleotides of the present invention can be labeled, if
desired, by
incorporating a label detectable by spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. For example, useful labels include
radioactive substances
(32P, 35S,3 H, 125 1), fluorescent dyes (5-bromodesoxvuridin, fluorescein,
acetylaminofluorene,
digoxigenin) or biotin. Preferably, polynucleotides are labeled at their 3'
and 5' ends. Examples
of non-radioactive labeling of nucleic acid fragments are described in the
French patent No. FR-
7810975 or by Urdea et al (1988) or Sanchez-Pescador et al (1988). In
addition, the probes
according to the present invention may have structural characteristics such
that they allow the
signal amplification, such structural characteristics being, for example,
branched DNA probes as
those described by Urdea et al. in 1991 or in the European patent No. EP 0 225
807 (Chiron), the
disclosures of which are incorporated by reference herein in their entirety.
A label can also be used to capture the primer, so as to facilitate the
immobilization of
either the primer or a primer extension product. such as amplified DNA, on a
solid support. A
capture label is attached to the primers or probes and can be a specific
binding member which
forms a binding pair with the solid's phase reagent's specific binding member
(e.g. biotin and
streptavidin). Therefore depending upon the type of label carried by a
polynucleotide or a probe,
it may be employed to capture or to detect the target DNA. Further, it will be
understood that
the polynucleotides, primers or probes provided herein, may, themselves, serve
as tiie capture
label. For example, in the case where a solid phase reagent's binding member
is a nucleic acid
sequence, it may be selected such that it binds a complementarv portion of a
primer or probe to
thereby immobilize the primer or probe to the solid phase. In cases where a
polvnucleotide
probe itself serves as the binding member, those skilled in the art will
reco~nize that the probe
will contain a sequence or''tail" that is not complementary to the target. In
the case where a
polynucleotide primer itself serves as the capture label, at least a portion
of the primer will be
free to hybridize with a nucleic acid on a solid phase. DNA Labeling
techniques are well known
to the skilled technician.
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The probes of the present invention are useful for a number of purposes. They
can be
notably used in Southern hybridization to genomic DNA. The probes can also be
used to detect
PCR amplification products. They may also be used to detect mismatches in the
purHgene or
mRNA using other techniques.
5 Any of the polynucleotides, primers and probes of the present invention can
be
conveniently immobilized on a solid support. Solid supports are known to those
skilled in the
art and include the walls of wells of a reaction tray, test tubes, polystyrene
beads, magnetic
beads, nitrocellulose strips, membranes, microparticles such as latex
particles, sheep (or other
animal) red blood cells, duracytes and others. The solid support is not
critical and can be
10 selected by one skilled in the art. Thus, latex particles, microparticles,
magnetic or non-
magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or
silicon chips, sheep
(or other suitable animal's) red blood cells and duracvtes are all suitable
examples. Suitable
methods for immobilizing nucleic acids on solid phases include ionic,
hydrophobic, covalent
interactions and the like. A solid support, as used herein, refers to any
material which is
15 insoluble, or can be made insoluble by a subsequent reaction. The solid
support can be chosen
for its intrinsic ability to attract and immobilize the capture reagent.
Alternatively, the solid
phase can retain an additional receptor which has the ability to attract and
immobilize the
capture reagent. The additional receptor can include a charged substance that
is oppositely
charged with respect to the capture reagent itself or to a charged substance
conjugated to the
20 capture reagent. As yet another alternative, the receptor molecule can be
any specific binding
member which is immobilized upon (attached to) the solid support and which has
the ability to
immobilize the capture reagent through a specific binding reaction. The
receptor molecule
enables the indirect binding of the capture reagent to a solid support
material before the
performance of the assay or during the performance of the assay. The solid
phase thus can be a
25 plastic, derivatized plastic, magnetic or non-magnetic metal, glass or
silicon surface of a test
tube, microtiter well, sheet, bead, microparticle. chip, sheep (or other
suitable animal's) red
blood cells, duracytes and other configurations known to those of ordinary
skill in the art. The
polynucleotides of the invention can be attached to or immobilized on a solid
support
individually or in groups of at least 2. 5. 8, 10. 12, 15. 20, or 25 distinct
polynucleotides of the
30 invention to a single solid support. In addition. polynucleotides other
than those of the invention
may be attached to the same solid support as one or more polynucleotides of
the invention.
Consequentlv. the invention also deals with a method for detecting the
presence of a
nucleic acid comprising a nucleotide sequence selected from a group consisting
of SEQ ID Nos
1, 2, a fragment or a variant thereof and a complementarv sequence tiiereto in
a sample, said
35 method comprising the following steps of:
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36
a) bringing into contact a nucleic acid probe or a plurality of nucleic acid
probes which
can hybridize with a nucleotide sequence included in a nucleic acid selected
from the
group consisting of the nucleotide sequences of SEQ ID Nos 1, 2, a fragment or
a
variant thereof and a complementary sequence thereto and the sample to be
assayed.
b) detecting the hybrid complex formed between the probe and a nucleic acid in
the
sample.
The invention further concerns a kit for detecting the presence of a nucleic
acid
comprising a nucleotide sequence selected from a group consisting of SEQ ID
Nos 1, 2, a
fragment or a variant thereof and a complementarv sequence thereto in a
sample, said kit
comprising:
a) a nucleic acid probe or a plurality of nucieic acid probes which can
hybridize with a
nucleotide sequence included in a nucleic acid selected form the group
consisting of
the nucleotide sequences of SEQ ID Nos 1, 2, a fragment or a variant thereof
and a
complementary sequence thereto;
b) optionally, the reagents necessary for performing the hybridization
reaction.
In a first preferred embodiment of this detection method and kit, said nucleic
acid probe
or the plurality of nucleic acid probes are labeled with a detectable
molecule. In a second
preferred embodiment of said method and kit, said nucleic acid probe or the
plurality of nucleic
acid probes has been immobilized on a substrate. In a third preferred
embodiment, the nucleic
acid probe or the plurality of nucleic acid probes comprise either a sequence
which is selected
from the group consisting of the nucleotide sequences of P1 to P42 and the
complementary
sequence thereto. B I to B34, C 1 to C34, D 1 to D42. E 1 to E42, or a
biallelic marker selected
from the group consisting of A 1 to A43 and the complements thereto.
Oligonucleotide Arrays
A substrate comprising a plurality of oligonucleotide primers or probes of the
invention
may be used either for detecting or amplifving targeted sequences in the pzrrH
gene and may also
be used for detecting mutations in the coding or in the non-coding sequences
of the purH gene.
Any polvnucleotide provided herein may be attached in overlapping areas or at
random
locations on the solid support. Alternatively the polynucleotides of the
invention may be
attached in an ordered array wherein each polynucleotide is attached to a
distinct region of the
solid support which does not overlap with the attachment site of any other
polynucleotide.
Preferably. such an ordered array of polvnucleotides is designed to be
"addressable" where the
distinct locations are recorded and can be accessed as part of an assay
procedure. Addressable
polynucleotide arrays typically comprise a plurality of different
oligonucleotide probes that are
CA 02368672 2001-09-24
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37
coupled to a surface of a substrate in different known locations. The
knowledge of the precise
location of each polynucleotides location makes these "addressable" arrays
particularly useful in
hybridization assays. Any addressable array technology known in the art can be
employed with
the polynucleotides of the invention. One particular embodiment of these
polynucleotide arrays
is known as the GenechipsTM, and has been generally described in US Patent
5,143,854; PCT
publications WO 90/15070 and 92/10092, the disclosures of which are
incorporated by reference
herein in their entirety. These arrays may generallv be produced using
mechanical synthesis
methods or light directed synthesis methods which incorporate a combination of
photolithographic methods and solid phase oligonucleotide synthesis (Fodor et
al., 1991). The
immobilization of arrays of oligonucleotides on solid supports has been
rendered possible by the
development of a technology generally identified as "Very Large Scale
Immobilized Polymer
Synthesis" (VLSIPST"') in which, typicallv, probes are immobilized in a high
density array on a
solid surface of a chip. Examples of VLSIPSTM technologies are provided in US
Patents
5,143,854; and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and
WO
95/1 1995, the disclosures of which are incorporated by reference herein in
their entirety, which
describe methods for forming oligonucleotide arrays through techniques such as
light-directed
synthesis techniques. In designing strategies aimed at providing arrays of
nucleotides
immobilized on solid supports, further presentation strategies were developed
to order and
display the oligonucleotide arrays on the chips in an attempt to maximize
hybridization patterns
and sequence information. Examples of such presentation strategies are
disclosed in PCT
Publications WO 94/12305. WO 94/11530, WO 97/29212 and WO 97/31256, the
disclosures of
which are incorporated by reference herein in their entirety.
In another embodiment of the oligonucleotide arrays of the invention, an
oligonucleotide
probe matrix may advantageously be used to detect mutations occurring in the
purHgene and
preferably in its regulatory region. For this particular purpose, probes are
specifically designed
to have a nucleotide sequence allowing their hybridization to the genes that
carry known
mutations (either by deletion. insertion or substitution of one or several
nucleotides). By known
mutations, it is meant, mutations on the pztrH gene that have been identified
according, for
example to the technique used by Huang et al.(1996) or Samson et al.(1996).
Another technique that is used to detect mutations in the purH gene is the use
of a high-
density DNA array. Each oligonucleotide probe constitutiilg a unit element of
the high density
DNA array is designed to match a specific subsequence of the purH genomic DNA
or cDNA.
Thus, an array consisting of oligonucleotides complementary to subsequences of
the target gene
sequence is used to determine the identity of the target sequence with the
wild gene sequence,
measure its amount, and detect differences between the target sequence and the
reference wild
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38
gene sequence of the purHgene. In one such design, termed 4L tiled array, is
implemented a set
of four probes (A, C, G, T), preferably 15-nucleotide oligomers. In each set
of four probes, the
perfect complement will hybridize more strongly than mismatched probes.
Consequently, a
nucleic acid target of length L is scanned for mutations with a tiled array
containing 4L probes.
the whole probe set containing all the possible mutations in the known wild
reference sequence.
The hybridization signals of the 15-mer probe set tiled array are perturbed by
a single base
change in the target sequence. As a consequence, there is a characteristic
loss of signal or a
"footprint" for the probes flanking a mutation position. This technique was
described by Chee et
al. in 1996, which is herein incorporated by reference.
Consequently, the invention concerns an array of nucleic acid molecules
comprising at
least one polynucleotide described above as probes and primers. Preferably,
the invention
concerns an array of nucleic acid comprising at least two polynucleotides
described above as
probes and primers.
A further object of the invention consists of an arrav of nucleic acid
sequences
comprising either at least one of the sequences selected from the group
consisting of Pl to P42,
B 1 to B34, C 1 to C34, D 1 to D42, El to E42, the sequences complementary
thereto, a fragment
thereof of at least 8, 10, 12, 15, 18, 20, 25, 30. or 40 consecutive
nucleotides thereof, and at least
one sequence comprising a biallelic marker selected from the group consisting
of A1 to A43 and
the complements thereto.
The invention also pertains to an array of nucleic acid sequences comprising
either at
least two of the sequences selected from the group consisting of P I to P42. B
1 to B34, C 1 to
C34, D I to D42, E l to E42, the sequences complementary thereto, a fragment
thereof of at least
8 consecutive nucleotides thereof, and at least two sequences comprising a
biallelic marker
selected from the group consisting of A 1 to A43 and the complements thereof.
purH Proteins and Polvpeptide Fraizments:
The term "purH polypeptides" is used herein to embrace all of the proteins and
polypeptides of the present invention. Also forming part of the invention are
polypeptides
encoded by the polynucleotides of the invention, as well as fusion
polypeptides comprising such
polypeptides.
The invention concerns the polypeptide encoded by a nucleotide sequence
selected from
the group consisting of SEQ ID No I or 2, a complenientarv sequence tliereof
or a fragment
thereto.
The invention embodies purH proteins from humans. including isolated or
purified purH
proteins consisting, consisting essentially, or comprising the sequence of SEQ
ID No 3. It
CA 02368672 2001-09-24
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39
should be noted the purH proteins of the invention are based on the naturally-
occurring variant
of the amino acid sequence of human purH, wherein the threonine residue of
amino acid position
116 has been replaced with a serine residue. This variant protein and the
fragments thereof
which contain a serine at the amino acid position 116 of SEQ ID No 3 are
collectively referred to
herein as "1 16-Ser variants."
The present invention embodies isolated, purified, and recombinant
polypeptides
comprising a contiguous span of at least 6 amino acids, preferably at least 8
to 10 amino acids,
more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ
ID No 3, wherein
said contiguous span includes a serine residue at amino acid position 116 in
SEQ ID No 3. In
other preferred embodiments the contiguous stretch of amino acids comprises
the site of a
mutation or functional mutation, including a deletion, addition, swap or
truncation of the amino
acids in the purH protein sequence.
purH proteins are preferably isolated from human or mammalian tissue samples
or
expressed from human or mammalian genes. The purH polypeptides of the
invention can be
made using routine expression methods known in the art. The polynucleotide
encoding the
desired polypeptide, is ligated into an expression vector suitable for any
convenient host. Both
eukaryotic and prokaryotic host systems is used in forming recombinant
polypeptides, and a
summary of some of the more common systems. The polypeptide is then isolated
from lysed
cells or from the culture medium and purified to the extent needed for its
intended use.
Purification is by any technique known in the art, for example, differential
extraction, salt
fractionation, chromatography, centrifugation, and the like. See, for example,
Methods in
Enzymology for a variety of methods for purifving proteins.
In addition, shorter protein fragments is produced by chemical synthesis.
Alternatively
the proteins of the invention is extracted from cells or tissues of humans or
non-human animals.
Methods for purifying proteins are known in the art, and include the use of
detergents or
chaotropic agents to disrupt particles followed by differential extraction and
separation of the
polypeptides by ion exchange chromatography, affinity chromatography,
sedimentation
according to density, and gel electrophoresis.
Any pzrrHcDNA, including SEQ ID No 2. is used to express purH proteins and
polypeptides. The nucleic acid encodin- the purH protein or polypeptide to be
expressed is
operably linked to a promoter in an expression vector using conventional
cloning technology. The
parrH insert in the expression vector may comprise the full coding sequence
for the purH protein or
a portion thereof. For example, the purH derived insert niav encode a
polypeptide comprising at
least 10 consecutive amino acids of the purH protein of SEQ ID No 3, wherein
said consecutive
amino acids comprising a serine residue in amino acid position 116.
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The expression vector is any of the mammalian, yeast, insect or bacterial
expression
systems known in the art. Commercially available vectors and expression
systems are available
from a variety of suppliers including Genetics Institute (Cambridge, MA),
Stratagene (La Jolla,
California), Promega (Madison, Wisconsin), and Invitrogen (San Diego,
California). If desired, to
5 enhance expression and facilitate proper protein folding, the codon context
and codon pairing of the
sequence is optimized for the particular expression organism in which the
expression vector is
introduced, as explained by Hatfield, et al., U.S. Patent No. 5,082,767, the
disclosures of which are
incorporated by reference herein in their entirety.
In one embodiment, the entire coding sequence of the purH cDNA through the
poly A
10 signal of the cDNA are operably linked to a promoter in the expression
vector. Alternatively, if the
nucleic acid encoding a portion of the purH protein lacks a methionine to
serve as the initiation site,
an initiating methionine can be introduced next to the first codon of the
nucleic acid using
conventional techniques. Similarly, if the insert from the purHcDNA lacks a
poly A signal, this
sequence can be added to the construct by, for example, splicing out the Poly
A signal from pSG5
15 (Stratagene) using Bgll and Sail restriction endonuclease enzymes and
incorporating it into the
mammalian expression vector pXTI (Stratagene). pXT I contains the LTRs and a
portion of the gag
gene from Moloney Murine Leukemia Virus. The position of the LTRs in the
construct allow
efficient stable transfection. The vector includes the Herpes Simplex
Thymidine Kinase promoter
and the selectable neomycin gene. The nucleic acid encoding the purH protein
or a portion thereof
20 is obtained by PCR from a bacterial vector containing the purHcDNA of SEQ
ID No 2 using
oligonucleotide primers complementary to the purHcDNA or portion thereof and
containing
restriction endonuclease sequences for Pst I incorporated into the 5'primer
and Bg1It at the 5' end of
the corresponding cDNA 3' primer, taking care to ensure that the sequence
encoding the purH
protein or a portion thereof is positioned properly with respect to the poly A
signal. The purified
25 fragment obtained front the resulting PCR reaction is digested with Pstl,
blunt ended with an
exonuclease, digested with Bgl II, purified and ligated to pXTI, now
containing a poly A signal and
digested with BglII.
The ligated product is transfected into mouse NIH 3T3 cells using Lipofectin
(Life
Technologies, Inc., Grand Island, New York) under conditions outlined in the
product specification.
30 Positive transfectants are selected after growing the transfected cells in
600ug/ml G41 8 (Sigma, St.
Louis. Missouri).
Alternatively, the nucleic acids encoding the purH protein or a portion
thereof is cloned into
pED6dpc2 (Genetics Institute. Cambridge, MA). The resulting pED6dpc2
constructs is transfected
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41
into a suitable host cell, such as COS 1 cells. Methotrexate resistant cells
are selected and
expanded.
The above procedures may also be used to express a mutant purH protein
responsible for a
detectable phenotype or a portion thereof.
The expressed proteins is purified using conventional purification techniques
such as
ammonium sulfate precipitation or chromatographic separation based on size or
charge. The protein
encoded by the nucleic acid insert may also be purified using standard
immunochromatography
techniques. In such procedures, a solution containing the expressed purH
protein or portion thereof,
such as a cell extract, is applied to a column having antibodies against the
purH protein or portion
thereof is attached to the chromatography matrix. The expressed protein is
allowed to bind the
immunochromatography column. Thereafter, the column is washed to remove non-
specifically
bound proteins. The specifically bound expressed protein is then released from
the column and
recovered using standard techniques.
To confirm expression of the purH protein or a portion thereof, the proteins
expressed from
host cells containing an expression vector containing an insert encoding the
purH protein or a
portion thereof can be compared to the proteins expressed in host cells
containing the expression
vector without an insert. The presence of a band in samples from cells
containing the expression
vector with an insert which is absent in samples from cells containing the
expression vector without
an insert indicates that the purH protein or a portion thereof is being
expressed. Generally, the band
will have the mobility expected for the purH protein or portion thereof.
However, the band may
have a mobility different than that expected as a result of modifications such
as glycosylation,
ubiquitination, or enzymatic cleavage.
Antibodies capable of specifically recognizing the expressed purH protein or a
portion
thereof are described below.
If antibody production is not possible, the nucleic acids encoding the purH
protein or a
portion thereof is incorporated into expression vectors designed for use in
purification schemes
employing chimeric polypeptides. In such strategies the nucleic acid encoding
the purH protein or a
portion thereof is inserted in frame with the gene encoding the other half of
the chimera. The other
half of the chimera is (3-globin or a nickel binding polypeptide encoding
sequence. A
chromatography matrix having antibody to (3-globin or nickel attached thereto
is then used to purify
the chimeric protein. Protease cleavage sites is engineered between the (3-
globin gene or the nickel
binding polypeptide and the purH protein or portion thereof. Thus, the two
polypeptides of the
chimera is separated from one another by protease digestion.
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42
One useful expression vector for generating (3-globin chimeric proteins is
pSG5
(Stratagene), which encodes rabbit (3-globin. Intron II of the rabbit (3-
globin gene facilitates splicing
of the expressed transcript, and the polyadenylation signal incorporated into
the construct increases
the level of expression. These techniques are well known to those skilled in
the art of molecular
biology. Standard methods are published in methods texts such as Davis et al.,
(1986) and many of
the methods are available from Stratagene, Life Technologies, Inc., or
Promega. Polypeptide may
additionally be produced from the construct using in vitro translation svstems
such as the In vitro
ExpressTM Translation Kit (Stratagene).
Antibodies That Bind purH Polvpeptides of the Invention
Any purH polypeptide or whole protein may be used to Qenerate antibodies
capable of
specifically binding to expressed purH protein or fraaments thereof as
described. The antibody
compositions of the invention are capable of specifically binding or
specifically bind to the 116-
Ser variant of the purH protein. For an antibody composition to specifically
bind to the 116-Ser
variant of purH it must demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%, or
100% greater
binding affinity for full lengtli I 16-Ser variant of purH than for full
length 116-Thr variant of
purH in an ELISA, RIA, or other antibody-based binding assay.
In a preferred embodiment of the invention antibody compositions are capable
of
selectively binding, or selectively bind to an epitope-containing fragment of
a polypeptide
comprising a contiguous span of at least 6 amino acids, preferably at least 8
to 10 amino acids,
more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ
ID No 3, wherein
said epitope comprises a serine residue at amino acid position 116 in SEQ ID
No 3, wherein said
antibodv composition is optionallv either polvclonal or monoclonal.
The present invention also contemplates the use of polypeptides comprising a
contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino
acids, more
preferably at least 12, 15, 20, 25, 50, or 100 amino acids of a purH
polypeptide in the
manufacture of antibodies, wherein said contiguous span comprises a serine
residue at amino
acid position 116 of SEQ ID No 3. In a preferred embodiment such polypeptides
are useful in
the manufacture of antibodies to detect the presence and absence of the 1 16-
Ser variant.
Non-huinan animals or mammals. whether wild-type or transgenic, which express
a
different species of purH than the one to wiiich antibody binding is desired,
and animals which
do not express purH (i.e. a purH knock out animal as described in herein) are
particularly useful
for preparing antibodies. purH knock out animals will i-ecognize all or most
of the exposed
regions of purH as foreign antigens, and therefore produce antibodies with a
wider array of purH
epitopes. Moreover, smaller polypeptides with onlv 10 to 30 amino acids may be
useful in
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43
obtaining specific binding to the I 16-Ser variant. In addition, the hu-noral
immune system of
animals which produce a species of purH that resembles the antigenic sequence
will
preferentially recognize the differences between the animal's native purH
species and the
antigen sequence, and produce antibodies to these unique sites in the antigen
sequence. Such a
technique will be particularly useful in obtaining antibodies that
specifically bind to the 116-Ser
variant.
Preparation of Antibody Compositions to the 116-SerVariant of purH
Substantially pure protein or polypeptide is isolated from transfected or
transformed cells
containing an expression vector encoding the purH protein or a portion
thereof. The concentration
of protein in the final preparation is adjusted, for example, by concentration
on an Amicon filter
device, to the level of a few microgams/ml. Monoclonal or polyclonal antibody
to the protein can
then be prepared as follows:
A. Monoclonal Antibody Production by Hvbridoma Fusion
Monoclonal antibody to epitopes in the purH protein or a portion thereof can
be prepared
from murine hybridomas according to the classical method of Kohler, G. and
Milstein, C., (1975)
or derivative methods thereof. Also see Harlow, E., and D. Lane. 1988.
Briefly, a mouse is repetitively inoculated with a few micrograms of the purH
protein or a
portion thereof over a period of a few weeks. The mouse is then sacrificed,
and the antibody
producing cells of the spleen isolated. The spleen cells are fused by means of
polyethylene glycol
with mouse myeloma cells, and the excess unfused cells destroyed by growth of
the system on
selective media comprising aminopterin (HAT media). The successfully fused
cells are diluted and
aliquots of the dilution placed in wells of a microtiter plate where growth of
the culture is continued.
Antibody-producing clones are identified by detection of antibody in the
supernatant fluid of the
wells by immunoassay procedures, sucli as ELISA, as originally described by
Engvall, E (1980),
and derivative methods thereof. Selected positive clones can be expanded and
their monoclonal
antibody product harvested for use. Detailed procedures for monoclonal
antibody production are
described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New
York. Section 21-
2.
B. Polvclonal Antibodv Production bv Immunization
Polyclonal antiserum containing antibodies to heterogeneous epitopes in the
purH protein or
a portion thereof can be prepared by immunizing suitable non-hLunan animal
with the purH protein
or a portion thereof, which can be unmodified or modified to enhance
immunogenicity. A suitable
non-human animal is preferablv a non-human mammal is selected. usually a
mouse, rat, rabbit,
goat, or horse. Alternatively. a crude preparation which has been enriched for
purH
concentration can be used to generate antibodies. Such proteins, fragments or
preparations are
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introduced into the non-human mammal in the presence of an appropriate
adjuvant (e.g.
aluminum hydroxide. RIBI, etc.) which is known in the art. In addition the
protein, fragment or
preparation can be pretreated with an agent which will increase antigenicity,
such agents are
known in the art and include, for example, methylated bovine serum albumin
(mBSA), bovine
serum albumin (BSA), Hepatitis B surface antigen, and keyhole limpet
hemocyanin (KLH).
Serum from the immunized animal is collected, treated and tested according to
known
procedures. If the serum contains polyclonal antibodies to undesired epitopes,
the polyclonal
antibodies can be purified by immunoaffinity chromatography.
Effective polyclonal antibody production is affected by many factors related
both to the
antigen and the host species. Also, host animals vary in response to site of
inoculations and
dose, with both inadequate or excessive doses of antigen resulting in low
titer antisera. Small
doses (ng level) of antigen administered at multiple intradermal sites appears
to be most reliable.
Techniques for producing and processing polyclonal antisera are known in the
art, see for
example, Mayer and Walker (1987). An effective immunization protocol for
rabbits can be
found in Vaitukaitis, J. et al. (1971).
Booster injections can be given at regular intervals, and antiserum harvested
when antibody
titer thereof, as determined semi-quantitatively, for example, by double
immunodiffusion in agar
against known concentrations of the antigen, begins to fall. See, for example,
Ouchterlony, O. et al.,
(1973). Plateau concentration of antibody is usually in the range of 0.1 to
0.2 mg/ml of serum
(about 12 M). Affinity of the antisera for the antigen is determined by
preparing competitive
binding curves, as described, for example, by Fisher. D., (1980).
Antibody preparations prepared according to either the monoclonal or the
polyclonal
protocol are useful in quantitative immunoassays which determine
concentrations of antigen-
bearing stibstances in biological samples; they are also used semi-
quantitatively or qualitatively to
identify the presence of antigen in a biological sample. The antibodies may
also be used in
therapeutic compositions for killing cells expressing the protein or reducing
the levels of the protein
in the body.
The antibodies of the invention may be labeled. either by a radioactive, a
fluorescent or an
enzymatic label.
Consequently, the invention is also directed to a metliod for detecting
specifically the
presence of a human purH polypeptide according to the invention in a
biological sample, said
method comprising the following steps:
a) bringing into contact the biological sample with a polyclonal or monoclonal
antibody
directed against the purH polypeptide of the amino acid sequence of SEQ ID No
3, or to a
peptide fragment or variant thereof:
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b) detecting the antigen-antibody complex formed.
The invention also concerns a diagnostic kit for detecting in vitro the
presence of a
human BAP28 polypeptide according to the present invention in a biological
sample, wherein
said kit comprises :
5 a) a polyclonal or monoclonal antibodv directed against the purH polypeptide
of the
amino acid sequence of SEQ ID No 3, or to a peptide fragment or variant
thereof, optionally
labeled:
b) a reagent allowing the detection of the antigen-antibody complexes formed,
said
reagent carrying optionally a label, or being able to be recognized itself by
a labeled reagent,
10 more particularly in the case when the above-mentioned monoclonal or
polyclonal antibody is
not labeled by itself.
In a preferred embodiment of the detection method and kit, the purH
polypeptide
comprises a Serine residue in position 1 16 of the SEQ ID No 3.
purH-related Bialielic Markers
15 Advantages Of The Biallelic Markers Of The Present Invention
The parrH-related biallelic markers of the present invention offer a number of
important
advantages over other genetic markers such as RFLP (Restriction fragment
length
polymorphism) and VNTR (Variable Number of Tandem Repeats) markers.
The first generation of markers, were RFLPs, which are variations that modify
the length
20 of a restriction fragment. But methods used to identify and to type RFLPs
are relatively wasteful
of materials, effort, and time. The second generation of genetic markers were
VNTRs, which
can be categorized as either minisatellites or microsatellites. Minisatellites
are tandemly
repeated DNA sequences present in units of 5-50 repeats which are distributed
along regions of
the human chromosomes ranging from 0.1 to 20 kilobases in length. Since they
present many
25 possible alleles, their informative content is very high. Minisatellites
are scored by performing
Southern blots to identify the number of tandem repeats present in a nucleic
acid sample from
4
the individual being tested. However, there are only 10 potential VNTRs that
can be typed by
Southern blotting. Moreover, both RFLP and VNTR markers are costly and time-
consuming to
develop and assay in large numbers.
30 SNP or biallelic markers can be used in the same manner as RFLPs and VNTRs
but
offer several advantages. SNP are densely spaced in the human genome and
represent the most
frequent type of variation. An estimated number of more than 10' sites are
scattered along the
3x109 base pairs of the human genome. Therefore. SNP occur at a(yreater
frequencv and with
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greater uniformity than RFLP or VNTR markers which means that there is a
greater probability
that such a marker will be found in close proximity to a genetic locus of
interest. SNP are less
variable than VNTR markers but are mutationally more stable.
Also, the different forms of a characterized single nucleotide polvmorphism.
such as the
biallelic markers of the present invention, are often easier to distinguish
and can therefore be
typed easily on a routine basis. Biallelic markers have single nucleotide
based alleles and they
have only two common alleles, which allows highly parallel detection and
automated scoring.
The biallelic markers of the present invention offer the possibility of rapid,
high throughput
genotyping of a large number of individuals.
Biallelic markers are densely spaced in the genome, sufficiently informative
and can be
assayed in large numbers. The combined effects of these advantages make
biallelic markers
extremely valuable in genetic studies. Biallelic markers can be used in
linkage studies in
families, in allele sharing methods, in linkage disequilibrium studies in
populations, in
association studies of case-control populations or of trait positive and trait
negative populations.
An important aspect of the present invention is that biallelic markers allow
association studies to
be performed to identify genes involved in complex traits. Association studies
examine the
frequency of marker alleles in unrelated case- and control-populations and are
generally
employed in the detection of polygenic or sporadic traits. Association studies
may be conducted
within the general population and are not limited to studies performed on
related individuals in
affected families (linkage studies). Biallelic markers in different genes can
be screened in
parallel for direct association with disease or response to a treatment. This
multiple gene
approach is a powerful tool for a variety of human genetic studies as it
provides the necessary
statistical power to examine the synergistic effect of multiple genetic
factors on a particular
phenotype, drug response, sporadic trait, or disease state with a complex
genetic etiology.
purH-Related Biallelic Markers And Polynucleotides Related Thereto
The invention also concerns purH-related biallelic markers. As used herein the
term
"purH-related biallelic marker" relates to a set of biallelic markers in
linkage disequilibrium
with the purH gene. The term purH-related biallelic marker includes the
biallelic markers
designated A 1 to A43.
A portion of the biallelic markers of the present invention are disclosed in
Table 2.
Their location on the purH gene is indicated in Table 2 and also as a single
base polymorphism
in the features of in the related SEQ ID Nos 1. 2. and 4 to 22. The pairs of
primers allowing the
amplification of a nucleic acid containing the polymorphic base of one purH
biallelic marker are
listed in Table I of Example 2.
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19purH-related biallelic markers, A1 to A17, A34 and A35, are located in the
genomic
sequence of purH. Two of them are located in exonic sequence, namely A2 and
A15. The
biallelic marker A2 provides an amino acid change in which a threonine residue
in position 116
of the protein sequence is replaced by a serine residue. 24 purH-related
biallelic markers, A18 to
A33 and A36 to A43, are located outside of the genomic sequence of purH.
However, there are
in linkage disequilibrium with thepurH gene. 12 of them, A 18 to A20, A26,
A28, A32, A33,
A39, A42 to A44, and A46, are located in intergenic regions. The others are
located in a gene
localized near the purH gene. This gene is the fibronectin gene.
In a preferred embodiment, the sequences comprising a polymorphic base of one
of the
biallelic markers listed in Table 2 are selected from the group consisting of
the nucleotide
sequences that have a contiguous span of, that consist of, that are comprised
in, or that comprises
a polynucleotide selected from the group consisting of the nucleic acids of
the sequences set
forth as the amplicons listed in Table I or a variant thereof or a
complementary sequence
thereto.
The invention further concerns a nucleic acid encoding the purH protein,
wherein said
nucleic acid comprises a polymorphic base of a biallelic marker selected from
the group
consisting of A1 to A17, A34 and A35 and the complements thereof.
The primers for amplification or sequencing reaction of a polynucleotide
comprising a
biallelic marker of the invention may be designed from the disclosed sequences
for any method
known in the art. A preferred set of primers are fashioned such that the 3'
end of the contiguous
span of identity with a sequence selected from the group consisting of SEQ ID
Nos 1, 2, and 4 to
22 or a sequence complementary thereto or a variant thereof is present at the
3' end of the
primer. Such a configuration allows the 3' end of the primer to hybridize to a
selected nucleic
acid sequence and dramatically increases the efficiency of the primer for
amplification or
sequencing reactions. Allele specific primers may be designed such that a
polymorphic base of a
biallelic marker is at the 3' end of the contiguous span and the contiguous
span is present at the 3'
end of the primer. Such allele specific primers tend to selectively prime an
amplification or
sequencing reaction so long as they are used with a nucleic acid sample that
contains one of the
two alleles present at a biallelic marker. The 3' end of the primer of the
invention may be
located within or at least 2, 4, 6, 8, 10, 12. 15, 18, 20. 25, 50, 100. 250,
500, or 1000 nucleotides
upstream of apurH-related biallelic marker in said sequence or at any other
location which is
appropriate for their intended use in sequencinti. amplification or the
location of novel sequences
or markers. Tlius, another set of preferred amplification primers comprise an
isolated
polvnucleotide consisting essentially of a contiguous span of 8 to 50
nucleotides in a sequence
selected from the aroup consisting of SEQ ID Nos 1, 2, and 4 to 22 or a
sequence
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complementary thereto or a variant thereof, wherein the 3' end of said
contiguous span is located
at the 3'end of said polynucleotide, and wherein the 3'end of said
polynucleotide is located
upstream of a purH-related biallelic marker in said sequence. Preferably,
those amplification
primers comprise a sequence selected from the group consisting of the
sequences B1 to B34 and
C1 to C34. Primers with their 3' ends located I nucleotide upstream of apurH-
related biallelic
marker have a special utility in microsequencing assays. Preferred
microsequencing primers are
described in Table 3. Optionally, microsequencing primers are selected from
the group
consisting of the nucleotide sequences D1 to D42 and E1 to E42.
The probes of the present invention may be designed from the disclosed
sequences for
any method known in the art, particularly methods which allow for testing if a
marker disclosed
herein is present. A preferred set of probes may be designed for use in the
hybridization assays
of the invention in any manner known in the art such that they selectively
bind to one aliele of a
biallelic marker, but not the other allele under any particular set of assay
conditions. Preferred
hybridization probes comprise the polymorphic base of either allele I or
allele 2 of the specific
biallelic marker. Optionally, said biallelic marker may be within 6, 5, 4, 3,
2, or I nucleotides of
the center of the hybridization probe or at the center of said probe.
It should be noted that the polynucleotides of the present invention are not
limited to
having the exact flanking sequences surrounding the polymorphic bases which
are enumerated in
Sequence Listing. Rather, it will be appreciated that the flanking sequences
surrounding the
biallelic markers may be lengthened or shortened to any extent compatible with
their intended
use and the present invention specifically contemplates such sequences. The
flanking regions
outside of the contiguous span need not be homologous to native flanking
sequences which
actually occur in human subjects. The addition of any nucleotide sequence
which is compatible
with the nucleotides intended use is specifically contemplated.
Primers and probes may be labeled or immobilized on a solid support as
described in
"Oligonucleotide probes and primers".
The polynucleotides of the invention which are attached to a solid support
encompass
polynucleotides with any further limitation described in this disclosure, or
those following,
specified alone or in any combination: Optionallv, said polynucleotides may be
specified as
attached individually or in groups of at least 2. 5. 8. 10, 12, 15, 20, or 25
distinct polynucleotides
of the invention to a single solid support. Optionally, polvnucleotides other
than those of the
invention may attached to the same solid support as polynucleotides of the
invention.
Optionally, when multiple polynucleotides are attached to a solid support they
may be attached
at random locations, or in an ordered array. Optionally, said ordered array
may be addressable.
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The present invention also encompasses diagnostic kits comprising one or more
polynucleotides of the invention with a portion or all of the necessary
reagents and instructions
for genotyping a test subject by determining the identity of a nucleotide at
aparrH-related
biallelic marker. The polynucleotides of a kit may optionally be attached to a
solid support, or
be part of an array or addressable array of polynucleotides. The kit may
provide for the
determination of the identity of the nucleotide at a marker position by any
method known in the
art including, but not limited to, a sequencing assay method, a
microsequencing assay method, a
hybridization assay method, or an enzyme-based mismatch detection method.
Optionally such a
kit may include instructions for scoring the results of the determination with
respect to the test
subjects' risk of suffering from a form of cancer or prostate cancer, the
level of aggressiveness of
cancer tumors or prostate cancer tumors, an early onset of cancer or prostate
cancer, a beneficial
response to or side effects related to treatment against cancer or prostate
cancer.
Methods For De Novo Identification Of Biallelic Markers
Any of a variety of methods can be used to screen a genomic fragment for
single
nucleotide polymorphisms such as differential hybridization with
oligonucleotide probes,
detection of changes in the mobility measured by gel electrophoresis or direct
sequencing of the
amplified nucleic acid. A preferred method for identifying biallelic markers
involves
comparative sequencing of genomic DNA fragments from an appropriate number of
unrelated
individuals.
In a first embodiment, DNA samples from unrelated individuals are pooled
together,
following which the genomic DNA of interest is amplified and sequenced. The
nucleotide
sequences thus obtained are then analvzed to identify significant
polymorphisms. One of the
major advantages of this method resides in the fact that the pooling of the
DNA samples
substantially reduces the number of DNA amplification reactions and sequencing
reactions,
which must be carried out. Moreover, this method is sufficiently sensitive so
that a biallelic
marker obtained thereby usually demonstrates a sufficient frequency of its
less common allele to
be useful in conducting association studies.
In a second embodiment, the DNA samples are not pooled and are therefore
amplified
and sequenced individually. This metliod is usually preferred when biallelic
markers need to be
identified in order to perform association studies within candidate genes.
Preferably, highly
relevant gene reoions such as promoter regions or exon regions may be screened
for biallelic
markers. A biallelic marker obtained using this method may show a lower degree
of
informativeness for conducting association studies, e.g. if the frequency of
its less frequent allele
may be less than about 10%. Such a biallelic marker will. however, be
sufficiently informative
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to conduct association studies and it will further be appreciated that
including less informative
biallelic markers in the genetic analysis studies of the present invention,
may allow in some
cases the direct identification of causal mutations, which may, depending on
their penetrance, be
rare mutations.
5 The following is a description of the various parameters of a preferred
method used by
the inventors for the identification of the biallelic markers of the present
invention.
Genomic DNA Samples
The genomic DNA samples from which the biallelic markers of the present
invention are
generated are preferably obtained from unrelated individuals corresponding to
a heterogeneous
10 population of known ethnic background. The number of individuals from whom
DNA samples
are obtained can vary substantially, preferably froin about 10 to about 1000,
preferably from
about 50 to about 200 individuals. It is usually preferred to collect DNA
samples from at least
about 100 individuals in order to have sufficient polymorphic diversity in a
given population to
identify as many markers as possible and to generate statistically significant
results.
15 As for the source of the genomic DNA to be subjected to analysis, any test
sample can
be foreseen without any particular limitation. These test samples include
biological samples,
which can be tested by the methods of the present invention described herein,
and include human
and animal body fluids such as whole blood, serum, plasma, cerebrospinal
fluid, urine, lymph
fluids, and various external secretions of the respiratory, intestinal and
genitourinary tracts, tears,
20 saliva, milk, white blood cells, myelomas and the like; biological fluids
such as cell culture
supernatants; fixed tissue specimens including tumor and non-tumor tissue and
lymph node
tissues; bone marrow aspirates and fixed cell specimens. The preferred source
of genomic DNA
used in the present invention is from peripheral venous blood of each donor.
Techniques to
prepare genomic DNA from biological samples are well known to the skilled
technician. Details
25 of a preferred embodiment are provided in Example 1. The person skilled in
the art can choose
to amplify pooled or unpooled DNA samples.
DNA Amplification
The identification of biallelic markers in a sample of genoinic DNA may be
facilitated
throutrh the use of DNA amplification methods. DNA samples can be pooled or
unpooled for
30 the ainplification step. DNA amplification techniques are well known to
those skilled in the art.
Amplification techniques that can be used in the context of the present
invention include,
but are not limited to, the ligase chain reaction (LCR) described in EP-A- 320
308, WO 9320227
and EP-A-439 182, the disclosures of which are incorporated herein by
reference, the
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polymerase chain reaction (PCR, RT-PCR) and techniques such as the nucleic
acid sequence
based amplification (NASBA) described in Guatelli J.C., et al.(1990) and in
Compton J.(1991),
Q-beta amplification as described in European Patent Application No 4544610,
strand
displacement amplification as described in Walker et al.(1996) and EP A 684
315 and, target
mediated amplification as described in PCT Publication WO 9322461, the
disclosures of which
are incorporated herein by reference in their entirety. For amplification of
mRNAs, it is within
the scope of the present invention to reverse transcribe mRNA into cDNA
followed by
polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps
as described in
U.S. Patent No. 5,322,770 or, to use Asymmetric Gap LCR (RT-AGLCR) as
described by
Marshall et al.(1994). AGLCR is a modification of GLCR that allows the
amplification of
RNA.
The PCR technology is the preferred amplification technique used in the
present
invention. A variety of PCR techniques are familiar to those skilled in the
art. For a review of
PCR technology, see White (1997) and the publication entitled "PCR Methods and
Applications" (1991, Cold Spring Harbor Laboratory Press). In each of these
PCR procedures,
PCR primers on either side of the nucleic acid sequences to be amplified are
added to a suitably
prepared nucleic acid sample along with dNTPs and a thermostable polymerase
such as Taq
polymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in the sample
is denatured
and the PCR primers are specifically hybridized to complementary nucleic acid
sequences in the
sample. The hybridized primers are extended. Tliereafter, another cycle of
denaturation,
hybridization, and extension is initiated. The cycles are repeated multiple
times to produce an
amplified fragment containing the nucleic acid sequence between the primer
sites. PCR has
further been described in several patents including US Patents 4,683,195;
4,683,202; and
4,965,188. Each of the preceding publications is incorporated herein by
reference in their
entirety.
The PCR technology is the preferred amplification technique used to identify
new
biallelic markers. A typical example of a PCR reaction suitable for the
purposes of the present
invention is provided in Example 2.
One of the aspects of the present invention is a method for the amplification
of the
human pairHgene, particularly of the genomic sequence of SEQ ID No I or of the
cDNA
sequence of SEQ ID No 2, or a fragment or a variant thereof in a test sample,
preferably using
the PCR technology. This method comprises the steps of contacting a test
sample suspected of
containing the targetpzrrHencoding sequence or portion thereof with
amplification reaction
reagents comprising a pair of amplification primers. and eventually in some
instances a detection
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probe that can hybridize with an internal region of amplicon sequences to
confirm that the
desired amplification reaction has taken place.
Thus, the present invention also relates to a method for the amplification of
a human purH
gene sequence, particularly of a portion of the genomic sequences of SEQ ID No
I or of the
cDNA sequence of SEQ ID No 2, or a variant thereof in a test sample, said
method comprising
the steps of:
a) contacting a test sample suspected of containing the targeted pzrrH gene
sequence
comprised in a nucleotide sequence selected from a group consisting of SEQ ID
Nos I
and 2, or fragments or variants thereof with amplification reaction reagents
comprising
a pair of amplification primers as described above and located on either side
of the
polynucleotide region to be amplified, and
b) optionally, detecting the amplification products.
The invention also concerns a kit for the amplification of a human purH gene
sequence,
particularly of a portion of the genomic sequence of SEQ ID No I or of the
cDNA sequence of
SEQ ID No 2, or a variant thereof in a test sample, wherein said kit
comprises:
a) a pair of oligonucleotide primers located on either side of the purH region
to be
amplified;
b) optionally, the reagents necessary for performing the amplification
reaction.
In one embodiment of the above amplification method and kit, the amplification
product
is detected by hybridization with a labeled probe having a sequence which is
complementary to
the amplified region. In another embodiment of the above amplification method
and kit, primers
comprise a sequence which is selected from the group consisting of the
nucleotide sequences of
B I to B34, C 1 to C34, D l to D42, and E I to E42.
In a first embodiment of the present invention, biallelic markers are
identified using
genomic sequence information generated by the inventors. Sequenced genomic DNA
fragments
are used to design primers for the amplification of 500 bp fragments. These
500 bp fragments
are amplified from genomic DNA and are scanned for biallelic markers. Primers
may be
designed using the OSP software (Hillier L. and Green P.. 1991). All primers
may contain,
upstream of the specific target bases, a common oligonucleotide tail that
serves as a sequencing
primer. Those skilled in the art are familiar with primer eYtensions, which
can be used for these
purposes.
Preferred primers, useful for the amplification of genomic sequences encoding
the
candidate genes, focus on promoters, exons and splice sites of the genes. A
biallelic marker
presetits a higher probability to be an evetitual causal mutation if it is
located in these functional
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regions of the gene. Preferred amplification primers of the invention include
the nucleotide
sequences B 1 to B34 and C 1 to C34, detailed further in Example 2, Table 1.
Sequencing Of Amplified Genomic DNA And Identification Of Single Nucleotide
Polymorphisms
The amplification products generated as described above, are then sequenced
using any
method known and available to the skilled technician. Methods for sequencing
DNA using
either the dideoxy-mediated method (Sanger method) or the Maxam-Gilbert method
are widely
known to those of ordinary skill in the art. Such methods are for example
disclosed in Sambrook
et al.(1989). Alternative approaches include hybridization to high-density DNA
probe arrays as
described in Chee et al.(1996).
Preferably, the amplified DNA is subjected to automated dideoxy terminator
sequencing
reactions using a dye-primer cycle sequencing protocol. The products of the
sequencing
reactions are run on sequencing gels and the sequences are determined using
gel image analysis.
The polymorphism search is based on the presence of superimposed peaks in the
electrophoresis
pattern resulting from different bases occurring at the same position. Because
each dideoxy
terminator is labeled with a different fluorescent molecule, the two peaks
corresponding to a
biallelic site present distinct colors corresponding to two different
nucleotides at the same
position on the sequence. However, the presence of two peaks can be an
artifact due to
background noise. To exclude such an artifact, the two DNA strands are
sequenced and a
comparison between the peaks is carried out. In order to be registered as a
polymorphic
sequence, the polymorphism has to be detected on botli strands.
The above procedure permits those amplification products, which contain
biallelic
markers to be identified. The detection limit for the frequency of biallelic
polymorphisms
detected by sequencing pools of 100 individuals is approximately 0.1 for the
minor allele, as
verified by sequencing pools of known allelic frequencies. However, more than
90% of the
biallelic polymorphisms detected by the pooling method have a frequency for
the minor allele
higher than 0.25. Therefore, the biallelic markers selected by this method
have a frequency of at
least 0.1 for the minor allele and less than 0.9 for the major allele.
Preferably at least 0.2 for the
minor allele and less than 0.8 for the major allele, more preferably at least
0.3 for the minor
allele and less than 0.7 for the major allele, thus a heterozygosity rate
higher than 0.18,
preferably higher than 0.32, more preferably higher than 0.42.
In another embodiment, biallelic markers are detected by sequencing individual
DNA
samples, the frequency of the minor allele of such a biallelic marker may be
less than 0.1.
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Validation Of The Biallelic Markers Of The Present Invention
The polymorphisms are evaluated for their usefulness as genetic markers by
validating
that both alleles are present in a population. Validation of the biallelic
markers is accomplished
by genotyping a group of individuals by a method of the invention and
demonstrating that both
alleles are present. Microsequencing is a preferred method of genotyping
alleles. The validation
by genotyping step may be performed on individual samples derived from each
individual in the
group or by genotyping a pooled sample derived from more than one individual.
The group can
be as small as one individual if that individual is heterozygous for the
allele in question.
Preferably the group contains at least three individuals, more preferably the
group contains five
or six individuals, so that a single validation test will be more likely to
result in the validation of
more of the biallelic markers that are being tested. It should be noted,
however, that when the
validation test is performed on a small group it may result in a false
negative result if as a result
of sampling error none of the individuals tested carries one of the two
alleles. Thus, the
validation process is less useful in demonstrating that a particular initial
result is an artifact, than
it is at demonstrating that there is a bona fide biallelic marker at a
particular position in a
sequence. All of the genotyping, haplotyping. association, and interaction
study methods of the
invention may optionally be performed solely with validated biallelic markers.
Evaluation Of The Frequency Of The Biallelic Markers Of The Present Invention
The validated biallelic markers are further evaluated for their usefulness as
genetic
markers by determining the frequency of the least common allele at the
biallelic marker site.
The higher the frequency of the less common allele the greater the usefulness
of the biallelic
marker is association and interaction studies. The determination of the least
common allele is
accomplished by genotyping a group of individuals by a method of the invention
and
demonstrating that both alleles are present. This determination of frequencv
by genotyping step
may be performed on individual samples derived from each individual in the
group or by
genotyping a pooled sample derived from more than one individual. The group
must be large
enough to be representative of the population as a hole. Preferably the group
contains at least
20 individuals, more preferably the group contains at least 50 individuals,
most preferably the
group contains at least 100 individuals. Of course the larger the group the
greater the accuracy
of the frequency determination because of reduced sampling error. For an
indication of the
frequency for the less common allele of a particular biallelic marker of the
invention see Figures
1 and 2. A biallelic marker wherein the frequencv of the less common allele is
30% or more is
termed a"high qualitv biallelic marker." All of the genotvping. haplotyping.
association, and
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interaction study methods of the invention may optionally be performed solely
with high quality
biallelic markers.
The invention also relates to methods of estimating the frequency of an allele
in a
population comprising: a) genotyping individuals from said population for said
biallelic marker
5 according to the method of the present invention; and b) determining the
proportional
representation of said biallelic marker in said population. In addition, the
methods of estimating
the frequency of an allele in a population of the invention encompass methods
with any further
limitation described in this disclosure, or those following, specified alone
or in any combination;
optionally, said purH-related biallelic marker is selected from the group
consisting of A 1 to A43,
10 and the complements thereof, or optionally the biallelic markers in linkage
disequilibrium
therewith; optionally, said purH-related biallelic marker is selected from the
group consisting of
A1. A3 to A14, A16 to A17, A34, and A35, and the complements thereof, or
optionally the
biallelic markers in linkage disequilibrium therewith: optionally, said purH-
related biallelic
marker is selected from the group consisting of A2 and A15, and the
complements thereof, or
15 optionally the biallelic markers in linkage disequilibrium therewith;
optionally, said purH-
related biallelic marker is selected from the group consisting of A 18 to A33
and A36 to A43;
optionally, wherein said purH-related biallelic marker is selected from the
group consisting of
A29, A7, A20, A 10 and A 13, and the complements thereof, or optionally the
biallelic markers in
linkage disequilibrium therewith; optionally, wherein said purH-related
biallelic marker is
20 selected from the group consisting of A30, A17, A28, A25, A21, and A14, and
the complements
thereof, or optionally the biallelic markers in linkage disequilibrium
therewith; Optionally,
determining the frequency of a biallelic marker allele in a population mav be
accomplished by
determining the identity of the nucleotides for both copies of said biallelic
marker present in the
genome of each individual in said population and calculating the proportional
representation of
25 said nucleotide at said purH-related biallelic marker for the population;
Optionally, determining
the proportional representation may be accomplished by performing a genotyping
method of the
invention on a pooled biological sample derived from a representative number
of individuals, or
each iiidividual, in said population, and calculating the proportional amount
of said nticleotide
compared with the total.
30 Methods For GenotvninZ An Individual For Biallelic Markers
Methods are provided to genotype a biological sample for one or more biallelic
markers
of the present invention, all of which may be performed in vitro. Such methods
of genotyping
comprise determining the identity of a nucleotide at a purH biallelic marker
site by any method
known in the art. These metliods find use in (Yenon ping case-control
populations in association
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studies as well as individuals in the context of detection of alleles of
biallelic markers which are
known to be associated with a given trait, in which case both copies of the
biallelic marker
present in individual's genome are determined so that an individual may be
classified as
homozygous or heterozygous for a particular allele.
These genotyping methods can be performed on nucleic acid samples derived from
a
single individual or pooled DNA samples.
Genotyping can be performed using similar methods as those described above for
the
identification of the biallelic markers, or using other genotyping methods
such as those further
described below. In preferred embodiments, the comparison of sequences of
amplified genomic
fragments from different individuals is used to identify new biallelic markers
whereas
microsequencing is used for genotyping known biallelic markers in diagnostic
and association
study applications.
In one embodiment the invention encompasses methods of genotyping comprising
determining the identity of a nucleotide at apurH-related biallelic marker or
the complement
thereof in a biological sample; optionally, said purH-related biallelic marker
is selected from the
group consisting of A1 to A43, and the complements thereof, or optionally the
biallelic markers
in linkage disequilibrium therewith; optionally, said purH-related biallelic
marker is selected
from the group consisting of Al, A3 to A14, A16 to A17, A34, and A35, and the
complements
thereof, or optionally the biallelic markers in linkage disequilibrium
therewith: optionally, said
purH-related biallelic marker is selected from the group consisting of A2 and
A15, and the
complements thereof, or optionally the biallelic markers in linkage
disequilibrium tlierewith;
optionally, said purH-related biallelic marker is selected from the group
consisting of A18 to
A33 and A36 to A43; optionally, wherein said purH-related biallelic marker is
selected from the
group consisting of A29, A7, A20, A 10 and A 13, and the complements thereof,
or optionally the
biallelic markers in linkage disequilibrium therewith; optionally, wherein
said purH-related
biallelic marker is selected from the group consisting of A30, A17, A28, A25,
A21, and A14,
and the complements thereof, or optionally the biallelic markers in linkage
disequilibrium
therewith; optionally, wherein said biological sample is derived from a single
subject; optionally,
wherein the identity of the nucleotides at said biallelic marker is determined
for botli copies of
said biallelic marker present in said individual's genome; optionally. wherein
said biological
sample is derived from multiple subjects; optionally, further comprising
amplif_ying a portion of
said sequence comprising the biallelic marker prior to said determining step:
optionally, wherein
said amplifying is performed by PCR; optionally, wherein said determinin, is
performed by a
hybridization assay, a sequencing assav, a microsequencing assav. or an enzyme-
based
niismatch detection assay.
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Source of Nucleic Acids for genotyping
Any source of nucleic acids, in purified or non-purified form, can be utilized
as the
starting nucleic acid, provided it contains or is suspected of containing the
specific nucleic acid
sequence desired. DNA or RNA may be extracted from cells, tissues, body fluids
and the like as
described above. While nucleic acids for use in the genotyping methods of the
invention can be
derived from any mammalian source, the test subjects and individuals from
which nucleic acid
samples are taken are generally understood to be human.
Amplification Of DNA Fragments Comprising Biallelic Markers
Methods and polynucleotides are provided to amplify a segment of nucleotides
comprising one or more biallelic marker of the present invention. It will be
appreciated that
amplification of DNA fragments comprising biallelic markers may be used in
various methods
and for various purposes and is not restricted to genotyping. Nevertheless,
many genotyping
methods, although not all, require the previous amplification of the DNA
region carrying the
biallelic marker of interest. Such methods specifically increase the
concentration or total
number of sequences that span the biallelic marker or include that site and
sequences located
either distal or proximal to it. Diagnostic assays may also rely on
amplification of DNA
segments carrying a biallelic marker of the present invention. Amplification
of DNA may be
achieved by any method known in the art. Amplification techniques are
described above in the
section entitled, "DNA amplification."
Some of these amplification methods are particularly suited for the detection
of single
nucleotide polymorphisms and allow the siniultaneous amplification of a target
sequence and the
identification of the polymorphic nucleotide as it is further described below.
The identification of biallelic markers as described above allows the design
of
appropriate oligonucleotides, which can be used as primers to amplify DNA
fragments
comprising the biallelic markers of the present invention. Amplification can
be performed using
the primers initially used to discover new biallelic markers which are
described herein or any set
of primers allowing the amplification of a DNA fragment comprising a biallelic
marker of the
present invention.
In some embodiments the present invention provides primers for amplifying a
DNA
fragment containing one or more biallelic markers of the present invention.
Preferred
amplification primers are listed in Example 2. It will be appreciated that the
primers listed are
merely exemplary and that anv other set of primers which produce amplification
products
containing one or more biallelic markers of the present invention are also of
use.
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The spacing of the primers determines the length of the segment to be
amplified. In the
context of the present invention, amplified segments carrying biallelic
markers can range in size
from at least about 25 bp to 35 kbp. Amplification fragments from 25-3000 bp
are typical,
fragments from 50-1000 bp are preferred and fragments from 100-600 bp are
highly preferred.
It will be appreciated that amplification primers for the biallelic markers
may be any sequence
which allow the specific amplification of any DNA fragment carrying the
markers.
Amplification primers may be labeled or immobilized on a solid support as
described in
"Oligonucleotide probes and primers".
Methods of Genotyping DNA samples for Biallelic Markers
Any method known in the art can be used to identify the nucleotide present at
a biallelic
marker site. Since the biallelic marker allele to be detected has been
identified and specified in
the present invention, detection will prove simple for one of ordinary skill
in the art by
employing any of a number of techniques. Many genotyping methods require the
previous
amplification of the DNA region carrying the biallelic marker of interest.
While the
amplification of target or signal is often preferred at present,
ultrasensitive detection methods
which do not require amplification are also encompassed by the present
genotyping methods.
Methods well-known to those skilled in the art that can be used to detect
biallelic
polymorphisms include methods such as, conventional dot blot analyzes, single
strand
conformational polymorphism analysis (SSCP) described by Orita et al.(1989),
denaturing
gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage
detection, and
other conventional techniques as described in Sheffield et al.(1991), White et
al.(1992), Grompe
et al.(1989 and 1993). Another method for determining the identity of the
nucleotide present at a
particular polymorphic site employs a specialized exonuclease-resistant
nucleotide derivative as
described in US patent 4,656,127, the disclosure of which is incorporated
herein by reference in
its entirety.
Preferred methods involve directly determinin-, the identity of the nucleotide
present at a
biallelic marker site by sequencing assay, enzyme-based mismatch detection
assay, or
hybridization assay. The following is a description of some preferred methods.
A highly
preferred method is the microsequencing technique. The term "sequencing" is
generallv used
herein to refer to polymerase extension of duplex priiner/template complexes
and includes both
traditional sequencing and microsequencing.;.
1) Sequencina Assays
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The nucleotide present at a polymorphic site can be determined bv sequencing
methods.
In a preferred embodiment, DNA samples are subjected to PCR amplification
before sequencing
as described above. DNA sequencing methods are described in "Sequencing Of
Amplified
Genomic DNA And Identification Of Single Nucleotide Polymorphisms".
Preferably, the amplified DNA is subjected to automated dideoxv terminator
sequencing
reactions using a dye-primer cycle sequencing protocol. Sequence analysis
allows the
identification of the base present at the biallelic marker site.
2) Microsequencing Assays
In microsequencing methods, the nucleotide at a polymorphic site in a target
DNA is
detected by a single nucleotide primer extension reaction. This method
involves appropriate
microsequencing primers which, hybridize just upstream of the polymorphic base
of interest in
the target nucleic acid. A polymerase is used to specifically extend the 3'
end of the primer with
one single ddNTP (chain terminator) complementary to the nucleotide at the
polymorphic site.
Next the identity of the incorporated nucleotide is determined in any suitable
way.
Typically, microsequencing reactions are carried out using fluorescent ddNTPs
and the
extended microsequencing primers are analyzed by electrophoresis on ABI 377
sequencing
machines to determine the identity of the incorporated nucleotide as described
in EP 412 883,
the disclosure of which is incorporated herein by reference in its entirety.
Alternatively capillary
electrophoresis can be used in order to process a higher number of assays
simultaneously. An
example of a typical microsequencing procedure that can be used in the context
of the present
invention is provided in Example 4.
Different approaches can be used for the labeling and detection of ddNTPs. A
homogeneous phase detection method based on fluorescence resonance energy
transfer has been
described by Chen and Kwok (1997) and Chen et al.(1997). In this method,
amplified genomic
DNA fragments containing polymorphic sites are incubated with a 5'-fluorescein-
labeled primer
in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and
a modified Taq
polymerase. The dye-labeled primer is extended one base by the dye-terminator
specific for the
allele present on the template. At the end of the genotyping reaction. the
fluorescence intensities
of the two dyes in the reaction mixture are analyzed directly without
separation or purification.
All these steps can be performed in the same tube and the fluorescence changes
can be
inonitored in real time. Alternatively, the extended primer mav be analvzed by
MALDI-TOF
Mass Spectrometry. The base at the polymorphic site is identified by the mass
added onto the
microsequencing primer (see Haff and Sinirnov, 1997).
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Microsequencing may be achieved by the established microsequencing method or
by
developments or derivatives thereof. Alternative methods include several solid-
phase
microsequencing techniques. The basic microsequencing protocol is the same as
described
previously, except that the method is conducted as a heterogeneous phase
assay, in which the
5 primer or the target molecule is immobilized or captured onto a solid
support. To simplify the
primer separation and the terminal nucleotide addition analysis,
oligonucleotides are attached to
solid supports or are modified in such ways that permit affinity separation as
well as polymerase
extension. The 5' ends and internal nucleotides of synthetic oligonucleotides
can be modified in
a number of different ways to permit different affinity separation approaches,
e.g., biotinylation.
10 If a single affinity group is used on the oligonucleotides, the
oligonucleotides can be separated
from the incorporated terminator regent. This eliminates the need of physical
or size separation.
More than one oligonucleotide can be separated from the terminator reagent and
analyzed
simultaneously if more than one affinity group is used. This permits the
analysis of several
nucleic acid species or more nucleic acid sequence information per extension
reaction. The
15 affinity group need not be on the priming oligonucleotide but could
alternatively be present on
the template. For example, immobilization can be carried out via an
interaction between
biotinvlated DNA and streptavidin-coated microtitration wells or avidin-coated
polystyrene
particles. In the same manner, oligonucleotides or templates may be attached
to a solid support
in a high-density format. In such solid phase microsequencing reactions,
incorporated ddNTPs
20 can be radiolabeled (Syvanen, 1994) or linked to fluorescein (Livak and
Hainer, 1994). The
detection of radiolabeled ddNTPs can be achieved through scintillation-based
techniques. The
detection of fluorescein-linked ddNTPs can be based on the binding of
antifluorescein antibody
conjugated with alkaline phosphatase, followed by incubation with a
chromogenic substrate
(such as p-nitrophenyl phosphate). Other possible reporter-detection pairs
include: ddNTP
25 linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate
(Harju et al., 1993)
or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with
o-
phenylenediamine as a substrate (WO 92/15712), the disclosure of which is
incorporated herein
by reference in its entirety. As yet another alternative solid-phase
microsequencing procedure,
Nyren et al.(1993) described a method relying on the detection of DNA
polvmerase activity by
30 an enzvmatic luminometric inorganic pyrophosphate detection assay (ELIDA).
Pastinen et al.(1997) describe a method for multiplex detection of single
nucleotide
polymorphism in which the solid phase minisequencing principle is applied to
an
oligonucleotide array format. High-density arrays of DNA probes attached to a
solid support
(DNA chips) are further described below.
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In one aspect the present invention provides polynucleotides and methods to
genotype
one or more biallelic markers of the present invention by performing a
microsequencing assay.
Preferred microsequencing primers include the nucleotide sequences Dl to D42
and El to E42.
It will be appreciated that the microsequencing primers listed in Example 4
are merely
exemplary and that, any primer having a 3' end immediately adjacent to the
polymorphic
nucleotide may be used. Similarly, it will be appreciated that microsequencing
analysis may be
performed for any biallelic marker or any combination of biallelic markers of
the present
invention. One aspect of the present invention is a solid support which
includes one or more
microsequencing primers listed in Example 4, or fragments comprising at least
8, 12, 15, 20, 25,
30, 40, or 50 consecutive nucleotides thereof, to the extent that such lengths
are consistent with
the primer described, and having a 3' terminus immediately upstream of the
corresponding
biallelic marker, for determining the identity of a nucleotide at a biallelic
marker site.
3) Mismatch detection assays based on polymerases and ligases
In one aspect the present invention provides polynucleotides and methods to
determine
the allele of one or more biallelic markers of the present invention in a
biological sample, by
mismatch detection assays based on polymerases and/or ligases. These assays
are based on the
specificity of polymerases and ligases. Polymerization reactions places
particularly stringent
requirements on correct base pairing of the 3' end of the amplification primer
and the joining of
two oligonucleotides hybridized to a target DNA sequence is quite sensitive to
mismatches close
to the ligation site, especially at the 3' end. Methods, primers and various
parameters to amplify
DNA fragments comprising biallelic markers of the present invention are
further described
above in "Amplification Of DNA Fragments Comprising Biallelic Markers".
Allele Specific Amplification Primers
Discrimination between the two alleles of a biallelic marker can also be
achieved by
allele specific amplification, a selective strategy, whereby one of the
alleles is amplified without
amplification of the other allele. This is accomplished by placing the
polymorphic base at the 3'
end of one of the amplification primers. Because the extension forms from the
3'end of the
primer, a mismatch at or near this position has an inhibitory effect on
amplification. Therefore,
under appropriate amplification conditions, these primers only direct
amplification on their
complementary allele. Determining the precise location of the mismatch and the
corresponding
assay conditions are well within the ordinary skill in the art.
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Ligation/Amplification Based Methods
The "Oligonucleotide Ligation Assay" (OLA) uses two oligonucleotides which are
designed to be capable of hybridizing to abutting sequences of a single strand
of a target
molecules. One of the oligonucleotides is biotinylated, and the other is
detectably labeled. If the
precise complementary sequence is found in a target molecule, the
oligonucleotides will
hybridize such that their termini abut, and create a ligation substrate that
can be captured and
detected. OLA is capable of detecting single nucleotide polymorphisms and may
be
advantageously combined with PCR as described by Nickerson et al.(1990). In
this method,
PCR is used to achieve the exponential amplification of target DNA, which is
then detected
using OLA.
Other amplification methods which are particularly suited for the detection of
single
nucleotide polymorphism include LCR (ligase chain reaction), Gap LCR (GLCR)
which are
described above in "Amplification of the purH gene". LCR uses two pairs of
probes to
exponentially amplify a specific target. The sequences of each pair of
oligonucleotides, is
selected to permit the pair to hybridize to abutting sequences of the same
strand of the target.
Such hybridization forms a substrate for a template-dependant ligase. In
accordance with the
present invention, LCR can be performed with oligonucleotides having the
proximal and distal
sequences of the same strand of a biallelic marker site. In one embodiment,
either
oligonucleotide will be designed to include the biallelic marker site. In such
an embodiment, the
reaction conditions are selected such that the oligonucleotides can be ligated
together only if the
target molecule either contains or lacks the specific nucleotide that is
complementary to the
biallelic marker on the oligonucleotide. In an alternative embodiment, the
oligonucleotides will
not include the biallelic marker, such that when they hybridize to the target
molecule, a "gap" is
created as described in WO 90/01069. This gap is then ''filled" with
complementary dNTPs (as
mediated by DNA polymerase), or by an additional pair of oligonucleotides.
Thus at the end of
each cycle, each single strand has a complement capable of serving as a target
during the next
cycle and exponential allele-specific amplification of the desired sequence is
obtained.
Ligase/Polymerase-mediated Genetic Bit AnalysisTM is another method for
determining
the identity of a nucleotide at a preselected site in a nucleic acid molecule
(WO 95/21271), the
disclosure of which is incorporated herein by reference in its entirety. This
method involves the
incorporation of a nucleoside triphosphate that is complementary to the
nucleotide present at the
preselected site onto the terminus of a primer molecule, and their subsequent
ligation to a second
oligonucleotide. The reaction is monitored by detecting a specific label
attached to the
reaction's solid phase or by detection in solution.
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4) Hybridization Assay Methods
A preferred method of determining the identity of the nucleotide present at a
biallelic
marker site involves nucleic acid hybridization. The hybridization probes,
which can be
conveniently used in such reactions, preferably include the probes defined
herein. Any
hybridization assay may be used including Southern hybridization, Northern
hybridization, dot
blot hybridization and solid-phase hybridization (see Sambrook et al., 1989).
Hybridization refers to the formation of a duplex structure by two single
stranded
nucleic acids due to complementary base pairing. Hybridization can occur
between exactly
complementary nucleic acid strands or between nucleic acid strands that
contain minor regions
of mismatch. Specific probes can be designed that hybridize to one form of a
biallelic marker
and not to the other and therefore are able to discriminate between different
allelic forms.
Allele-specific probes are often used in pairs, one member of a pair showing
perfect match to a
target sequence containing the original allele and the other showing a perfect
match to the target
sequence containing the alternative allele. Hybridization conditions should be
sufficiently
stringent that there is a significant difference in hybridization intensity
between alleles, and
preferably an essentially binary response, whereby a probe hybridizes to only
one of the alleles.
Stringent, sequence specific hybridization conditions, under which a probe
will hybridize only to
the exactly complementary target sequence are well known in the art (Sambrook
et al., 1989).
Stringent conditions are sequence dependent and will be different in different
circumstances.
Generally, stringent conditions are selected to be about 5 C lower than the
thermal melting point
(Tm) for the specific sequence at a defined ionic strength and pH. Although
such hybridization
can be performed in solution, it is preferred to employ a solid-phase
hybridization assay. The
target DNA comprising a biallelic marker of the present invention may be
amplified prior to the
hybridization reaction. The presence of a specific allele in the sample is
determined by detecting
the presence or the absence of stable hybrid duplexes formed between the probe
and the target
DNA. The detection of hybrid duplexes can be carried out by a number of
methods. Various
detection assay formats are well known which utilize detectable labels bound
to either the target
or the probe to enable detection of the hybrid duplexes. Typically,
hybridization duplexes are
separated from unhybridized nucleic acids and the labels bound to the duplexes
are then
detected. Those skilled in the art will recognize that wash steps may be
employed to wash away
excess target DNA or probe as well as unbound conjugate. Further, standard
heterogeneous
assay forinats are suitable for detecting the hybrids using the labels present
on the primers and
probes.
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Two recently developed assays allow hybridization-based allele discrimination
with no
need for separations or washes (see Landegren U. et al., 1998). The TaqMan
assay takes
advantage of the 5' nuclease activity of Taq DNA polymerase to digest a DNA
probe annealed
specifically to the accumulating amplification product. TaqMan probes are
labeled with a
donor-acceptor dye pair that interacts via fluorescence energy transfer.
Cleavage of the TaqMan
probe by the advancing polymerase during amplification dissociates the donor
dye from the
quenching acceptor dye, greatly increasing the donor fluorescence. All
reagents necessary to
detect two allelic variants can be assembled at the beginning of the reaction
and the results are
monitored in real time (see Livak et al., 1995). In an alternative homogeneous
hybridization
based procedure, molecular beacons are used for allele discriminations.
Molecular beacons are
hairpin-shaped oligonucleotide probes that report the presence of specific
nucleic acids in
homogeneous solutions. When they bind to their targets they undergo a
conformational
reorganization that restores the fluorescence of an internally quenched
fluorophore (Tyagi et al.,
1998).
The polynucleotides provided herein can be used to produce probes which can be
used in
hybridization assays for the detection of biallelic marker alleles in
biological samples. These
probes are characterized in that they preferably comprise between 8 and 50
nucleotides, and in
that they are sufficiently complementary to a sequence comprising a biallelic
marker of the
present invention to hybridize thereto and preferably sufficiently specific to
be able to
discriminate the targeted sequence for only one nucleotide variation. A
particularly preferred
probe is 25 nucleotides in length. Preferably the biallelic marker is within 4
nucleotides of the
center of the polynucleotide probe. In particularly preferred probes, the
biallelic marker is at the
center of said polynucleotide. Preferred probes comprise a nucleotide sequence
selected from
the group consisting of amplicons listed in Table 1 and the sequences
complementary thereto, or
a fragment thereof, said fragment comprising at least about 8 consecutive
nucleotides, preferably
10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and
containing a
polymorphic base. In preferred embodiments the polymorphic base(s) are within
5, 4, 3, 2, 1,
nucleotides of the center of the said polynucleotide. more preferably at the
center of said
polynucleotide.
Preferably the probes of the present invention are labeled or immobilized on a
solid
support. Labels and solid supports are further described in "Oligonucleotide
Probes and
Primers". The probes can be non-extendable as described in "Oligonucleotide
Probes and
Primers".
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By assaying the hybridization to an allele specific probe, one can detect the
presence or
absence of a biallelic marker allele in a given sample. High-Throughput
parallel hybridization in
array format is specifically encompassed within "hybridization assays" and are
described below.
5) Hybridization To Addressable Arrays Of Oligonucleotides
5 Hybridization assays based on oligonucleotide arrays rely on the differences
in
hybridization stability of short oligonucleotides to perfectly matched and
mismatched target
sequence variants. Efficient access to polymorphism information is obtained
through a basic
structure comprising high-density arrays of oligonucleotide probes attached to
a solid support
(e.g., the chip) at selected positions. Each DNA chip can contain thousands to
millions of
10 individual synthetic DNA probes arranged in a grid-like pattern and
miniaturized to the size of a
dime.
The chip technology has already been applied with success in numerous cases.
For
example, the screening of mutations has been undertaken in the BRCA I gene, in
S. cerevisiae
mutant strains, and in the protease gene of HIV-1 virus (Hacia et al., 1996;
Shoemaker et al.,
15 1996; Kozal et al., 1996). Chips of various formats for use in detecting
biallelic polymorphisms
can be produced on a customized basis by Affymetrix (GeneChipTM), Hyseq
(HyChip and
HyGnostics), and Protogene Laboratories.
In general, these methods employ arrays of oligonucleotide probes that are
complementary to target nucleic acid sequence segments from an individual
which, target
20 sequences include a polymorphic marker. EP 785280, the disclosure of which
is incorporated
herein by reference in its entirety. describes a tiling strategy for the
detection of single nucleotide
polymorphisms. Briefly, arrays may generally be "tiled" for a large number of
specific
polymorphisms. By "tiling" is generally meant the synthesis of a defined set
of oligonucleotide
probes which is made up of a sequence compiementary to the target sequence of
interest, as well
25 as preselected variations of that sequence, e.g., substitution of one or
more given positions with
one or more members of the basis set of nucleotides. Tiling strategies are
further described in
PCT application No. WO 95/11995, the disclosure of which is incorporated
herein by reference
in its entirety. In a particular aspect, arrays are tiled for a number of
specific, identified biallelic
marker sequences. In particular, the array is tiled to include a number of
detection blocks, each
30 detection block being specific for a specific biallelic marker or a set of
biallelic markers. For
example, a detection block may be tiled to include a number of probes, which
span the sequence
segment that includes a specific polymorphism. To ensure probes that are
complementary to
each allele, the probes are synthesized in pairs differing at the biallelic
marker. In addition to the
probes differing at the polymorphic base, monosubstituted probes are also
generally tiled within
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the detection block. These monosubstituted probes have bases at and up to a
certain number of
bases in either direction from the polymorphism, substituted with the
remaining nucleotides
(selected from A, T, G, C and U). Typically the probes in a tiled detection
block will include
substitutions of the sequence positions up to and including those that are 5
bases away from the
biallelic marker. The monosubstituted probes provide internal controls for the
tiled array, to
distinguish actual hybridization from artefactual cross-hybridization. Upon
completion of
hybridization with the target sequence and washing of the array, the array is
scanned to
determine the position on the array to which the target sequence hybridizes.
The hybridization
data from the scanned array is then analyzed to identify which allele or
alleles of the biallelic
marker are present in the sample. Hybridization and scanning may be carried
out as described in
PCT application No. WO 92/10092 and WO 95/11995 and US patent No. 5,424,186,
the
disclosures of which are incorporated by reference herein in their entirety.
Thus, in some embodiments, the chips may comprise an array of nucleic acid
sequences
of fragments of about 15 nucleotides in length. In further embodiments, the
chip may comprise
an array including at least one of the sequences selected from the group
consisting of amplicons
listed in table I and the sequences complementary thereto, or a fragment
thereof, said fragment
comprising at least about 8 consecutive nucleotides, preferably 10, 15, 20,
more preferably 25,
30, 40, 47, or 50 consecutive nucleotides and containing a polymorphic base.
In preferred
embodiments the polymorphic base is within 5, 4, 3, 2, 1, nucleotides of the
center of the said
polynucleotide, more preferably at the center of said polynucleotide. In some
embodiments, the
chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these
polynucleotides of the
invention. Solid supports and polynucleotides of the present invention
attached to solid
supports are further described in "oligonucleotide probes and primers".
6) Integrated Svstems
Another technique, which may be used to analyze polymorphisms, includes
multicomponent integrated systems, which miniaturize and compartmentalize
processes such as
PCR and capillary electrophoresis reactions in a single functional device. An
example of such
technique is disclosed in US patent 5.589,136. the disclosures of which are
incorporated by
reference herein in their entirety, wliich describes the integration of PCR
amplification and
capillary electrophoresis in chips.
Integrated systems can be envisaged mainly when microfluidic systems are used.
These
systems comprise a pattern of microchannels designed onto a glass, silicon,
quartz, or plastic
wafer included on a microchip. The movements of the samples are controlled by
electric,
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electroosmotic or hydrostatic forces applied across different areas of the
microchip to create
functional microscopic valves and pumps with no moving parts.
For genotyping biallelic markers, the microfluidic system mav integrate
nucleic acid
amplification, microsequencing, capillary electrophoresis and a detection
method such as laser-
induced fluorescence detection.
Methods Of Genetic Analysis Usin2 The Biallelic Markers Of The Present
Invention
Different methods are available for the Qenetic analysis of complex traits
(see Lander
and Schork, 1994). The search for disease-susceptibility genes is conducted
using two main
methods: the linkage approach in which evidence is sought for cosegregation
between a locus
and a putative trait locus using family studies. and the association approach
in which evidence is
sought for a statistically significant association between an allele and a
trait or a trait causing
allele (Khoury et al., 1993). In general, the biallelic markers of the present
invention find use in
any method known in the art to demonstrate a statistically significant
correlation between a
genotype and a phenotype. The biallelic markers may be used in parametric and
non-parametric
linkage analysis methods. Preferably, the biallelic markers of the present
invention are used to
identify genes associated with detectable traits using association studies. an
approach wliich does
not require the use of affected families and which permits the identification
of genes associated
with complex and sporadic traits.
The genetic analysis using the biallelic markers of the present invention may
be
conducted on any scale. The whole set of bial lelic markers of the present
invention or any
subset of biallelic markers of the present invention corresponding to the
candidate gene may be
used. Further. any set of (lenetic markers including a biallelic marker of the
present invention
may be used. A set of biallelic polymorphisms that could be used as genetic
markers in
combination with the biallelic markers of the present invention has been
described in WO
98/20165, the disclosure of which is incorporated herein by reference in its
entirety. As
mentioned above, it should be noted that the biallelic markers of the present
invention may be
included in anv complete or partiai genetic map of the human genome. These
different uses are
specificallv contemplated in the present invention and claims.
The invention also comprises methods of detecting an association between a
genotype
and a phenotype, comprising the steps of a) determining the frequency of at
least one purH-
related biallelic marker in a trait positive population according to
a;enotvping method of the
invention: b) determining the frequency of said pin-H-related biallelic marker
in a control
population according to a genotvping method of the invention: and c)
determining whether a
statistically significant association exists bemeen said aenotype and said
phenotype. In
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addition, the methods of detecting an association between a genotype and a
phenotype of the
invention encompass methods with any further limitation described in this
disclosure, or those
following, specified alone or in any combination: Optionally, said purH-
related biallelic marker
is selected from the group consisting of A 1 to A43, and the complements
thereof, or optionally
the biallelic markers in linkage disequilibrium therewith; optionally, said
purH-related biallelic
marker is selected from the group consisting of A1. A3 to A14, Al6 to A17,
A34, and A35, and
the complements thereof, or optionallv the biallelic markers in linkage
disequilibrium therewith;
optionally, said purH-related biallelic marker is selected from the group
consisting of A2 and
A15, and the complements thereof. or optionally the biallelic markers in
linkage disequilibrium
therewith; optionally, said purH-related biallelic marker is selected from the
group consisting of
A18 to A33 and A36 to A43; Optionally, said control population may be a trait
negative
population, or a random population; Optionally, each of said genotyping steps
a) and b) may be
performed on a pooled biological sample derived from each of said populations;
Optionally,
each of said genotyping of steps a) and b) is performed separately on
biological samples derived
from each individual in said population or a subsample thereof; Optionally,
said phenotype is
symptoms of, or susceptibility to prostate cancer, the level of aggressiveness
of prostate cancer
tumors, an early onset of prostate cancer, a beneficial response to or side
effects related to
treatment against prostate cancer.
The invention also encompasses methods of estimating the frequency of a
haplotype for
a set of biallelic markers in a population, comprising the steps of: a)
genotyping at least one
purH-related biallelic marker according to a method of the invention for each
individual in said
population: b) genotyping a second biallelic marker by determining the
identity of the
nucleotides at said second biallelic marker for both copies of said second
biallelic marker present
in the genome of each individual in said population; and c) applying a
haplotype determination
method to the identities of the nucleotides determined in steps a) and b) to
obtain an estimate of
said frequency. In addition, the methods of estimating the frequency of a
haplotype of the
invention encompass methods with any further limitation described in this
disclosure, or those
following, specified alone or in any combination: Optionally, saidpzrrH-
related biallelic marker
is selected from the group consisting of A1 to A43, and the comple-nents
thereof, or optionally
the biallelic markers in linkage disequilibrium therewith; optionally,
saidpurH-related biallelic
marker is selected from the group consisting of A 1, A3 to A 14, A 16 to A 17,
A34, and A35, and
the complements thereof, or optionally the bialielic markers in linkage
disequilibrium tlierewith;
optionally, said pan-H-related biallelic marker is selected from the group
consisting of A2 and
A15, and the complements thereof, or optionallv the biallelic markers in
Iinkage disequilibrium
therewith: optionally, said pznH related biallelic marker is selected from the
'~roup consisting of
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A18 to A33 and A36 to A43; Optionally, said haplotype determination method is
performed by
asymmetric PCR amplification, double PCR amplification of specific alleles,
the Clark
algorithm, or an expectation-maximization algorithm.
An additional embodiment of the present invention encompasses methods of
detecting
an association between a haplotype and a phenotype, comprising the steps of:
a) estimating the
frequency of at least one haplotype in a trait positive population, according
to a method of the
invention for estimating the frequency of a haplotype; b) estimating the
frequency of said
haplotype in a control population, according to a method of the invention for
estimating the
frequency of a haplotype; and c) determining whether a statistically
significant association exists
between said haplotype and said phenotype. In addition, the methods of
detecting an association
between a haplotype and a phenotype of the invention encompass methods with
any further
limitation described in this disclosure, or those following: Optionally, said
purH-related biallelic
marker is selected from the group consisting of A1 to A43, and the complements
thereof, or
optionally the biallelic markers in linkage disequilibrium therewith;
optionally, said purH-
related biallelic marker is selected from the group consisting of Al, A3 to
A14, A16 to A17,
A34, and A35, and the complements thereof, or optionally the biallelic markers
in linkage
disequilibrium therewith; optionally, said purH-related biallelic marker is
selected from the
group consisting of A2 and A15, and the complements thereof, or optionally the
biallelic
markers in linkage disequilibrium therewith; optionally, said purH-related
biallelic marker is
selected from the group consisting of A18 to A33 and A36 to A43; Optionally,
said control
population is a trait negative population, or a random population. Optionally,
said phenotype is
symptoms of. or susceptibility to prostate cancer, the level of aggressiveness
of prostate cancer
tumors, an early onset of prostate cancer, a beneficial response to or side
effects related to
treatment against prostate cancer; Optionally, said method comprises the
additional steps of
determining the phenotype in said trait positive and said control populations
prior to step c).
Linkage Analysis
Linkage analysis is based upon establishing a correlation between the
transmission of
genetic markers and that of a specific trait throughout generations within a
family. Thus, the aim
of linkage analysis is to detect marker loci that show cosegregation with a
trait of interest in
pedigrees.
Parametric Methods
When data are available from successive generations there is the opportunity
to study
the degree of linkage between pairs of loci. Estimates of the recombination
fraction enable loci
to be ordered and placed onto a genetic map. With loci that are genetic
markers, a genetic map
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can be established, and then the strength of linkage between markers and
traits can be calculated
and used to indicate the relative positions of markers and genes affecting
those traits (Weir,
1996). The classical method for linkage analysis is the logarithm of odds
(lod) score method
(see Morton, 1955; Ott, 1991). Calculation of lod scores requires
specification of the mode of
5 inheritance for the disease (parametric method). Generally, the length of
the candidate region
identified using linkage analysis is between 2 and 20Mb. Once a candidate
region is identified
as described above, analysis of recombinant individuals using additional
markers allows further
delineation of the candidate region. Linkage analysis studies have generally
relied on the use of
a maximum of 5,000 microsatellite markers, thus limiting the maximum
theoretical attainable
10 resolution of linkage analysis to about 600 kb on average.
Linkage analysis has been successfully applied to map simple genetic traits
that show
clear Mendelian inheritance patterns and which have a high penetrance (i.e.,
the ratio between
the number of trait positive carriers of allele a and the total number of a
carriers in the
population). However, parametric linkage analysis suffers from a variety of
drawbacks. First, it
15 is limited by its reliance on the choice of a genetic model suitable for
each studied trait.
Furthermore, as already mentioned, the resolution attainable using linkage
analysis is limited,
and complementary studies are required to refine the analysis of the typical
2Mb to 20Mb
regions initially identified through linkage analysis. In addition, parametric
linkage analysis
approaches have proven difficult when applied to complex genetic traits, such
as those due to the
20 combined action of multiple genes and/or environmental factors. It is very
difficult to model
these factors adequately in a lod score analysis. In such cases, too large an
effort and cost are
needed to recruit the adequate number of affected families required for
applying linkage analysis
to these situations, as recently discussed by Risch, N. and Merikangas, K.
(1996).
Non-Parametric Methods
25 The advantage of the so-called non-parametric methods for linkage analysis
is that they
do not require specification of the mode of inheritance for the disease, thev
tend to be more
useful for the analysis of complex traits. In non-parametric methods, one
tries to prove that the
inheritance pattern of a chromosomal region is not consistent with random
Mendelian
segregation by showing that affected relatives inherit identical copies of the
region more often
30 than expected by chance. Affected relatives should show excess ''allele
sharing" even in the
presence of incomplete penetrance and polygenic inheritance. In non-parametric
linkage
analysis the degree of agreement at ainarker locus in two individuals can be
measured either by
the number of alleles identical by state (IBS) or by the number of alleles
identical bv descent
(IBD). Affected sib pair analysis is a well-known special case and is the
simplest form of these
35 methods.
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The biallelic markers of the present invention may be used in both parametric
and non-
parametric linkage analysis. Preferably biallelic markers may be used in non-
parametric
methods which allow the mapping of genes involved in complex traits. The
biallelic markers of
the present invention may be used in both IBD- and IBS- methods to map genes
affecting a
complex trait. In such studies, taking advantage of the high density of
biallelic markers, several
adjacent biallelic marker loci may be pooled to achieve the efficiency
attained by multi-allelic
markers (Zhao et al., 1998).
Population Association Studies
The present invention comprises methods for identifying if the purH gene is
associated
with a detectable trait using the biallelic markers of the present invention.
In one embodiment
the present invention comprises methods to detect an association between a
biallelic marker
allele or a biallelic marker haplotype and a trait. Further, the invention
comprises methods to
identify a trait causing allele in linkage disequilibrium with any biallelic
marker allele of the
present invention.
As described above, alternative approaches can be employed to perform
association
studies: genome-wide association studies, candidate region association studies
and candidate
gene association studies. In a preferred embodiment, the biallelic markers of
the present
invention are used to perform candidate gene association studies. The
candidate gene analysis
clearly provides a short-cut approach to the identification of genes and gene
polymorphisms
related to a particular trait when some information concerning the biology of
the trait is
available. Further, the biallelic markers of the present invention may be
incorporated in any map
of genetic markers of the human genome in order to perform genome-wide
association studies.
Methods to generate a high-density map of biallelic markers has been described
in US
Provisional Patent application serial number 60/082,614. The biallelic markers
of the present
invention may further be incorporated in any map of a specific candidate
region of the genome
(a specific chromosome or a specific chromosomal segment for example).
As mentioned above, association studies may be conducted within the general
population and are not limited to studies performed on related individuals in
affected families.
Association studies are extremely valuable as they permit the analysis of
sporadic or multifactor
traits. Moreover, association studies represent a powerful method for fine-
scale mapping
enabling much finer mapping of trait causing alleles than linkage studies.
Studies based on
pedigrees often only narrow the location of the trait causing allele.
Association studies using the
biallelic markers of the present invention can therefore be used to refine the
location of a trait
causing allele in a candidate region identified by Linkage Analvsis metliods.
Moreover, once a
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chromosome segment of interest has been identified, the presence of a
candidate gene such as a
candidate gene of the present invention, in the region of interest can provide
a shortcut to the
identification of the trait causing allele. Biallelic markers of the present
invention can be used to
demonstrate that a candidate gene is associated with a trait. Such uses are
specifically
contemplated in the present invention.
Determining The Frequency Of A Biallelic Marker Allele Or Of A Biallelic
Marker
Haplotype In A Population
Association studies explore the relationships among frequencies for sets of
alleles
between loci.
Determining The Frequency Of An Allele In A Population
Allelic frequencies of the biallelic markers in a populations can be
determined using one
of the methods described above under the heading "Methods for genotyping an
individual for
biallelic markers", or any genotyping procedure suitable for this intended
purpose. Genotyping
pooled samples or individual samples can determine the frequency of a
biallelic marker aliele in
a population. One way to reduce the number of genotypings required is to use
pooled samples.
A major obstacle in using pooled samples is in terms of accuracy and
reproducibility for
determining accurate DNA concentrations in setting up the pools. Genotyping
individual
samples provides higher sensitivity, reproducibility and accuracy and; is the
preferred method
used in the present invention. Preferably, each individual is genotyped
separately and simple
gene counting is applied to determine the frequency of an allele of a
biallelic marker or of a
genotype in a given population.
Determining The Frequency Of A Haplotype In A Population
The gametic phase of haplotypes is unknown when diploid individuals are
heterozygous
at more than one locus. Using genealogical information in families gametic
phase can
sometimes be inferred (Perlin et al., 1994). When no genealogical information
is available
different strategies may be used. One possibility is that the multiple-site
heterozygous diploids
can be eliminated from the analysis, keeping only the homozygotes and the
single-site
heterozygote individuals, but this approach might lead to a possible bias in
the satnple
composition and the underestimation of low-frequency haplotypes. Another
possibility is that
single chromosomes can be studied independently, for example, by asymmetric
PCR
amplification (see Newton et al, 1989; Wu et al., 1989) or by isolation of
single chromosome by
limit dilution followed by PCR amplification (see Ruano et al., 1990).
Further, a sample may be
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haplotyped for sufficiently close biallelic markers by double PCR
amplification of specific
alleles (Sarkar, G. and Sommer S. S., 1991). These approaches are not entirely
satisfying either
because of their technical complexity, the additional cost they entail, their
lack of generalization
at a large scale, or the possible biases they introduce. To overcome these
difficulties, an
algorithm to infer the phase of PCR-amplified DNA genotypes introduced by
Clark, A.G.(1990)
may be used. Briefly, the principle is to start filling a preliminary list of
haplotypes present in
the sample by examining unambiguous individuals, that is, the complete
homozygotes and the
single-site heterozygotes. Then other individuals in the same sample are
screened for the
possible occurrence of previously recognized haplotypes. For each positive
identification, the
complementary haplotype is added to the list of recognized haplotypes, until
the phase
information for all individuals is either resolved or identified as
unresolved. This method
assigns a single haplotype to each multiheterozygous individual, whereas
several haplotypes are
possible when there are more than one heterozygous site. Alternatively, one
can use methods
estimating haplotype frequencies in a population without assigning haplotypes
to each
individual. Preferably, a method based on an expectation-maximization (EM)
algorithm
(Dempster et al., 1977) leading to maximum-likelihood estimates of haplotype
frequencies under
the assumption of Hardy-Weinberg proportions (random mating) is used (see
Excoffier L. and
Slatkin M., 1995). The EM algorithm is a generalized iterative maximum-
likelihood approach to
estimation that is useful when data are ambiguous and/or incomplete. The EM
algorithm is used
to resolve heterozygotes into haplotypes. Haplotype estimations are further
described below
under the heading "Statistical Methods." Any other method known in the art to
determine or to
estimate the frequency of a haplotype in a population may be used.
Linkage Disequilibrium Analysis
Linkage disequilibrium is the non-random association of alleles at two or more
loci and
represents a powerful tool for mapping genes involved in disease traits (see
Ajioka R.S. et al.,
1997). Biallelic markers, because they are densely spaced in the human genome
and can be
genotyped in greater numbers than other types of genetic markers (such as RFLP
or VNTR
markers), are particularly useful in genetic analysis based on linkage
disequilibrium.
When a disease mutation is first introduced into a population (by a new
mutation or the
immigration of a mutation carrier), it necessarily resides on a single
chromosome and thus on a
single "background" or''ancestral" haplotype of linked markers. Consequently,
there is
complete disequilibrium between these markers and the disease mutation: one
finds the disease
mutation only in the presence of a specific set of marker alleles. Through
subsequent
generations recombination events occur between the disease mutation and these
marker
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74
polymorphisms, and the disequilibrium gradually dissipates. The pace of this
dissipation is a
function of the recombination frequency, so the markers closest to the disease
gene will manifest
higher levels of disequilibrium than those that are further away. When not
broken up by
recombination, "ancestral" haplotypes and linkage disequilibrium between
marker alleles at
different loci can be tracked not only through pedigrees but also through
populations. Linkage
disequilibrium is usually seen as an association between one specific allele
at one locus and
another specific allele at a second locus.
The pattern or curve of disequilibrium between disease and marker loci is
expected to
exhibit a maximum that occurs at the disease locus. Consequently, the amount
of linkage
disequilibrium between a disease allele and closely linked genetic markers may
yield valuable
information regarding the location of the disease gene. For fine-scale mapping
of a disease
locus, it is useful to have some knowledge of the patterns of linkage
disequilibrium that exist
between markers in the studied region. As mentioned above the mapping
resolution achieved
through the analysis of linkage disequilibrium is much higher than that of
linkage studies. The
high density of biallelic markers combined with linkage disequilibrium
analysis provides
powerful tools for fine-scale mapping. Different methods to calculate linkage
disequilibrium are
described below under the heading "Statistical Methods".
Population-Based Case-Control Studies Of Trait-Marker Associations
As mentioned above, the occurrence of pairs of specific alleles at different
loci on the
same chromosome is not random and the deviation from random is called linkage
disequilibrium. Association studies focus on population frequencies and rely
on the
phenomenon of linkage disequilibrium. If a specific allele in a given gene is
directly involved in
causing a particular trait, its frequency will be statistically increased in
an affected (trait positive)
population, when compared to the frequency in a trait negative population or
in a random control
population. As a consequence of the existence of linkage disequilibrium, the
frequency of all
other alleles present in the haplotype carrying the trait-causing allele will
also be increased in
trait positive individuals compared to trait negative individuals or random
controls. Therefore,
association between the trait and any allele (specifically a biallelic marker
a(lele) in linkage
disequilibrium with the trait-causing allele will suffice to suggest the
presence of a trait-related
gene in that particular region. Case-control populations can be genotyped for
biallelic markers
to identifv associations that narrowly locate a trait causing allele. As any
marker in linkage
disequilibrium with one given marker associated with a trait will be
associated with the trait.
Linkage disequilibrium allows the relative frequencies in case-control
populations of a limited
number of genetic polvmorphisms (specifically biallelic markers) to be
analyzed as an
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alternative to screening all possible functional polymorphisms in order to
find trait-causing
alleles. Association studies compare the frequency of marker alleles in
unrelated case-control
populations, and represent powerful tools for the dissection of complex
traits.
Case-Control Populations (Inclusion Criteria)
5 Population-based association studies do not concern familial inheritance but
compare the
prevalence of a particular genetic marker, or a set of markers, in case-
control populations. They
are case-control studies based on comparison of unrelated case (affected or
trait positive)
individuals and unrelated control (unaffected, trait negative or random)
individuals. Preferably
the control group is composed of unaffected or trait negative individuals.
Further, the control
10 group is ethnically matched to the case population. Moreover, the contirol
group is preferably
matched to the case-population for the main known confusion factor for the
trait under study (for
example age-matched for an age-dependent trait). Ideally, individuals in the
two samples are
paired in such a way that they are expected to differ only in their disease
status. The terms "trait
positive population", "case population" and "affected population" are used
interchangeably
15 herein.
An important step in the dissection of complex traits using association
studies is the
choice of case-control populations (see Lander and Schork, 1994). A major step
in the choice of
case-control populations is the clinical definition of a given trait or
phenotype. Any genetic trait
may be analyzed by the association method proposed here by carefully selecting
the individuals
20 to be included in the trait positive and trait negative phenotypic groups.
Four criteria are often
useful: clinical phenotype, age at onset, family history and severity. The
selection procedure for
continuous or quantitative traits (such as blood pressure for example)
involves selecting
individuals at opposite ends of the phenotype distribution of the trait under
study, so as to
include in these trait positive and trait negative populations individuals
with non-overlapping
25 phenotypes. Preferably, case-control populations consist of phenotypically
homogeneous
populations. Trait positive and trait negative populations consist of
phenotypically uniform
populations of individuals representing each between I and 98%, preferably
between 1 and 80%,
more preferably between I and 50%, and more preferably between I and 30%, most
preferably
between I and 20% of the total population under study, and preferably selected
among
30 individuals exhibiting non-overlapping phenotypes. The clearer the
difference between the two
trait phenotypes, the greater the probability of detecting an association with
biallelic markers.
The selection of those drastically different but relatively uniform phenotypes
enables efficient
comparisons in association studies and the possible detection of marked
differences at the
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genetic level, provided that the sample sizes of the populations under study
are significant
enough.
In preferred embodiments, a first group of between 50 and 300 trait positive
individuals,
preferably about 100 individuals, are recruited according to their phenotypes.
A similar number
of control individuals are included in such studies.
In the present invention, typical examples of inclusion criteria include
prostate cancer.
Association Analysis
The general strategy to perform association studies using biallelic markers
derived from
a region carrying a candidate gene is to scan two groups of individuals (case-
control
populations) in order to measure and statistically compare the allele
frequencies of the biallelic
markers of the present invention in both groups.
If a statistically significant association with a trait is identified for at
least one or more of
the analyzed biallelic markers, one can assume that: either the associated
allele is directly
responsible for causing the trait (i.e. the associated allele is the trait
causing allele), or more
likely the associated allele is in linkage disequilibrium with the trait
causing allele. The specific
characteristics of the associated allele with respect to the candidate gene
function usually give
further insight into the relationship between the associated allele and the
trait (causal or in
linkage disequilibrium). If the evidence indicates that the associated allele
within the candidate
gene is most probably not the trait causing allele but is in linkage
disequilibrium with the real
trait causing allele, then the trait causing allele can be found by sequencing
the vicinity of the
associated marker, and performing further association studies with the
polymorphisms that are
revealed in an iterative manner.
Association studies are usually run in two successive steps. In a first phase,
the
frequencies of a reduced number of biallelic markers from the candidate gene
are determined in
the trait positive and control populations. In a second phase of the analysis,
the position of the
genetic loci responsible for the given trait is further refined using a higher
density of markers
from the relevant region. However, if the candidate gene under study is
relatively small in
length, as is the case for purH, a single phase may be sufficient to establish
significant
associations.
Haplotvpe Analvsis
As described above, when a chromosome carrying a disease allele first appears
in a
population as a result of either mutation or migration, the mutant allele
necessarily resides on a
chromosome having a set of linked markers: the ancestral haplotype. This
haplotype can be
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tracked through populations and its statistical association with a given trait
can be analyzed.
Complementing single point (allelic) association studies with multi-point
association studies also
called haplotype studies increases the statistical power of association
studies. Thus, a haplotype
association study allows one to define the frequency and the type of the
ancestral carrier
haplotype. A haplotype analysis is important in that it increases the
statistical power of an
analysis involving individual markers.
In a first stage of a haplotype frequency analysis, the frequency of the
possible
haplotypes based on various combinations of the identified biallelic markers
of the invention is
determined. The haplotype frequency is then compared for distinct populations
of trait positive
and control individuals. The number of trait positive individuals, which
should be, subjected to
this analysis to obtain statistically significant results usually ranges
between 30 and 300, with a
preferred number of individuals ranging between 50 and 150. The same
considerations apply to
the number of unaffected individuals (or random control) used in the studv.
The results of this
first analysis provide haplotype frequencies in case-control populations, for
each evaluated
haplotype frequency a p-value and an odd ratio are calculated. If a
statistically significant
association is found the relative risk for an individual carrying the given
haplotype of being
affected with the trait under study can be approximated.
Interaction AnalYsis
The biallelic markers of the present invention may also be used to identify
patterns of
biallelic markers associated with detectable traits resulting from polygenic
interactions. The
analysis of genetic interaction between alleles at unlinked loci requires
individual genotyping
using the techniques described herein. The analysis of allelic interaction
among a selected set of
biallelic markers with appropriate level of statistical significance can be
considered as a
haplotype analvsis. Interaction analysis consists in stratifying the case-
control populations with
respect to a given haplotype for the first loci and performing a haplotype
analysis with the
second loci with each subpopulation.
Statistical methods used in association studies are further described below.
Testing For Linkage In The Presence Of Association
The biallelic markers of the present invention may further be used in TDT
(transmission/disequilibrium test). TDT tests for both linkage and association
and is not affected
by population stratification. TDT requires data for affected individuals and
their parents or data
from unaffected sibs instead of from parents (see Spielmann S. et al., 1993;
Schaid D.J. et al.,
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1996, Spielmann S. and Ewens W.J., 1998). Such combined tests generally reduce
the false -
positive errors produced by separate analyses.
Statistical methods
In general, any method known in the art to test whether a trait and a genotype
show a
statistically significant correlation may be used.
1) Methods In Linkage Analysis
Statistical methods and computer programs useful for linkage analysis are well-
known to
those skilled in the art (see Terwilliger J.D. and Ott J., 1994; Ott J.,
1991).
2) Methods To Estimate Haplotype Frequencies In A Population
As described above, when genotypes are scored, it is often not possible to
distinguish
heterozygotes so that haplotype frequencies cannot be easily inferred. When
the gametic phase
is not known, haplotype frequencies can be estimated from the multilocus
genotypic data. Any
method known to person skilled in the art can be used to estimate haplotype
frequencies (see
Lange K., 1997; Weir, B.S., 1996) Preferably, maximum-Iikelihood haplotype
frequencies are
computed using an Expectation- Maximization (EM) algorithm (see Dempster et
al., 1977;
Excoffier L. and Slatkin M., 1995). This procedure is an iterative process
aiming at obtaining
maximum-likelihood estimates of haplotype frequencies from multi-locus
genotype data when
the gametic phase is unknown. Haplotype estimations are usually performed by
applying the
EM algorithm using for example the EM-HAPLO program (Hawley M. E. et al.,
1994) or the
Arlequin program (Schneider et al.. 1997). The EM algorithm is a generalized
iterative
maximum likelihood approach to estimation and is briefly described below.
Please note that in the present section. "Methods To Estimate Haplotype
Frequencies In
A Population, " of this text, phenotypes will refer to multi-locus genotypes
with unknown phase.
Genotypes will refer to known-phase multi-locus genotypes.
A sample of N unrelated individuals is typed for K markers. The data observed
are the
unknown-phase K-locus phenotypes that can categorized in F different
phenotypes. Suppose
that we have H underlying possible haplotypes (in case of K biallelic markers,
H=2'').
For phenotype j, suppose that cj genotypes are possible. We thus have the
following
equation
c. c.
ci J
Pi pr(genotype; )pr(hk , hl ) Equation I
i=1 i=1
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where Pj is the probability of the phenotypej, hk and hi are the two
haplotypes
constituent the genotype i. Under the Hardy-Weinberg equilibrium, pr(hk,hd
becomes:
pr(/ik, hl)= pr(hk )2 if hk = hl, Pr(/tk,/1/ )= 2 pr(hk ). pr(h1) if hk # hl.
Equation 2
The successive steps of the E-M algorithm can be described as follows:
Starting with initial values of the of haplotypes frequencies, noted p,( o) ,
p2o1 . pH>
these initial values serve to estimate the genotype frequencies (Expectation
step) and then
estimate another set of haplotype frequencies (Maximization step), noted pi'),
p;'~...... py'~
these two steps are iterated until changes in the sets of haplotypes frequency
are very small.
A stop criterion can be that the maximum difference between haplotype
frequencies
between two iterations is less than 10-'. These values can be adjusted
according to the desired
precision of estimations.
At a given iteration s, the Expectation step consists in calculating the
genotypes
frequencies by the following equation:
pr(genotype; )(S) = pr( phenotype j). pr(genotype; pltenotype j)(S)
n j pr(/ik /il)(s) Equation 3
= N p(S)
I
where genotype i occurs in phenotypej, and where hk and h, constitute genotype
i. Each
probability is derived according to eq. 1, and eq. 2 described above.
Then the Maximization step simply estimates another set of haplotype
frequencies given
the genotypes frequencies. This approach is also known as the gene-counting
method (Smith,
1957).
s+l 1 F c' s
Pt ) _ ~ Y Z~it =Pr(genotypel )() Eguation 4
2 j=1 i=1
Where (5it is an indicator variable which count the number of time haplotype t
in
genotype i. It takes the values of 0, 1 or 2.
To ensure that the estimation finally obtained is the maximum-likelihood
estimation
several values of departures are required. The estimations obtained are
compared and if they are
different the estimations leading to the best likelihood are kept.
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3) Methods To Calculate Linkage Disequilibrium Between Markers
A number of methods can be used to calculate linkage disequilibrium between
any two
genetic positions, in practice linkage disequilibrium is measured by applying
a statistical
association test to haplotype data taken from a population.
5 Linkage disequilibrium between any pair of biallelic markers comprising at
least one of
the biallelic markers of the present invention (M;, M) having alleles (a;/b;)
at marker M; and
alleles (aj/bj) at marker Mj can be calculated for every allele combination
(ai,aj, a;,bj, b;,aj and
b;,b), according to the Piazza formula:
Aaiaj= 404 - 4 (04 + 03) (04 +02), where:
10 04= -- = frequency of genotypes not having allele a; at M; and not having
allele aj at Mj
03= - += frequency of genotypes not having allele a; at M; arid having allele
aj at M;
02= + - = frequency of genotypes having allele a; at M; and not having allele
a; at M;
Linkage disequilibrium (LD) between pairs of biallelic markers (M;, Mj) can
also be
15 calculated for every allele combination (ai,aj; ai,bj; b;,aj and b;,b),
according to the maximum-
likelihood estimate (MLE) for delta (the composite genotypic disequilibrium
coefficient), as
described by Weir (Weir B. S., 1996). The MLE for the composite linkage
disequilibrium is:
Daiaj= (2ni + n) + n3 + n4/2)/N - 2(pr(ac)= pr(aj))
Where ni = E phenotype (a;/a;, a,/aj), n, = E phenotype (a;/a;, a/b), n3= E
phenotype
20 (a;/b;, aj/a;), n4= E phenotype (a;/b;, aj/bj) and N is the number of
individuals in the sample.
This formula allows linkage disequilibrium between alleles to be estimated
when only
genotype, and not haplotype, data are available.
Another means of calculating the linkage disequilibrium between markers is as
follows.
25 For a couple of biallelic markers, M, (a;/b;) and M; (a;/b;), fitting the
Hardy-Weinberg
equilibrium, one can estimate the four possible haplotype frequencies in a
given population
according to the approach described above.
The estimation of gametic disequilibrium between ai and aj is simply:
Daiaj = Pr(haplotyPe(ai , a j)) - Pr(oi ). pr(a j).
30 Where pr(a,) is the probability of allele a, and pr(a;) is the probability
of allele a; and
where pr(haplotvpe (a,, ci;)) is estimated as in Equation 3 above.
For a couple of biallelic marker only one ineasure of disequilibrium is
necessary to
describe the association between M, and M,.
Then a normalized value of the above is calculated as follows:
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D'aiaj = Daiaj / max (-pr(ai). pr(aj),-Pr(b+)= Pr(bj)) with Daiaj<0
D'aiaj = Daiaj / max (pr(bi)= pr(aj) , pr(ai)= pr(bj)) with Daia;>0
The skilled person will readily appreciate that other linkage disequilibrium
calculation
methods can be used.
Linkage disequilibrium among a set of biallelic markers having an adequate
heterozygosity rate can be determined by genotyping between 50 and 1000
unrelated
individuals, preferably between 75 and 200, more preferably around 100.
4) Testing For Association
Methods for determining the statistical significance of a correlation between
a phenotype
and a genotype, in this case an allele at a biallelic marker or a haplotype
made up of such alleles,
may be determined by any statistical test known in the art and with any
accepted threshold of
statistical significance being required. The appiication of particular methods
and thresholds of
significance are well with in the skill of the ordinary practitioner of the
art.
Testing for association is performed by determining the frequency of a
biallelic marker
allele in case and control populations and comparing these frequencies with a
statistical test to
determine if their is a statistically significant difference in frequency
which would indicate a
correlation between the trait and the biallelic marker allele under study.
Similarly, a haplotype
analysis is performed by estimating the frequencies of all possible haplotypes
for a given set of
biallelic markers in case and control populations, and comparing these
frequencies with a
statistical test to determine if their is a statistically significant
correlation between the haplotype
and the phenotype (trait) under study. Any statistical tool useful to test for
a statistically
significant association between a genotype and a phenotype may be used.
Preferably the
statistical test employed is a chi-square test with one degree of freedom. A P-
value is calculated
(the P-value is the probability that a statistic as large or larger than the
observed one would occur
by chance).
Statistical Sianificance
In preferred embodiments, significance for diagnosis purposes, either as a
positive basis
for further diacrnostic tests or as a preliminary starting point for early
preventive therapy, the p
value related to a biallelic marker association is preferably about I x 10-7
or less, more preferably
about 1 x 10--' or less, for a single biallelic marker analysis and about I x
10-' or less, still more
preferably I x 10-6 or less and most preferably of about I x 10-8 or less. for
a haplotype analysis
involving two or more markers. Tiiese values are believed to be applicable to
any association
studies involvinQ single or multiple marker combinations.
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The skilled person can use the range of values set forth above as a starting
point in order
to carry out association studies with biallelic markers of the present
invention. In doing so,
significant associations between the biallelic markers of the present
invention and prostate
cancer, the level of aggressiveness of prostate cancer tumors, an early onset
of prostate cancer,
or a beneficial response to or side effects related to treatment against
prostate cancer can be
revealed and used for diagnosis and drug screening purposes.
Phenotypic Permutation
In order to confirm the statistical significance of the first stage haplotype
analysis
described above, it might be suitable to perform further analyses in which
genotyping data from
case-control individuals are pooled and randomized with respect to the trait
phenotype. Each
individual genotyping data is randomly allocated to two groups, which contain
the same number
of individuals as the case-control populations used to compile the data
obtained in the first stage.
A second stage haplotype analysis is preferably run on these artificial
groups, preferably for the
markers included in the haplotype of the first stage analysis showing the
highest relative risk
coefficient. This experiment is reiterated preferablv at least between 100 and
10000 times. The
repeated iterations allow the determination of the probability to obtain by
chance the tested
haplotype.
Assessment Of Statistical Association
To address the problem of false positives similar analysis may be performed
with the
same case-control populations in random genomic regions. Results in random
regions and the
candidate region are compared as described in a co-pending US Provisional
Patent Application
entitled "Methods, Software And Apparati For Identifying Genomic Regions
Harboring A Gene
Associated With A Detectable Trait," U.S. Serial Number 60/107,986, filed
November 10,
1998, the contents of which are incorporated herein by reference.
5) Evaluation Of Risk Factors
The association between a risk factor (in genetic epidemiology the risk factor
is the
presence or the absence of a certain allele or haplotype at marker loci) and a
disease is measured
by the odds ratio (OR) and bv the relative risk (RR). If P(R-) is the
probability of developing the
disease for individuals with R and P(R-) is the probability for individuals
witliout the risk factor,
then the relative risk is simply the ratio of the two probabilities, that is:
RR= P(R )/P(R-)
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In case-control studies, direct measures of the relative risk cannot be
obtained because of
the sampling design. However, the odds ratio allows a good approximation of
the relative risk
for low-incidence diseases and can be calculated:
F+ ]/[ F-
OR =
1-F' (1-F-)
OR= (F+/(1-F'))/(F-/(1-F-))
F+ is the frequency of the exposure to the risk factor in cases and F- is the
frequency of
the exposure to the risk factor in controls. F and F- are calculated using the
allelic or haplotype
frequencies of the study and further depend on the underlying genetic model
(dominant,
recessive, additive...).
One can further estimate the attributable risk (AR) which describes the
proportion of
individuals in a population exhibiting a trait due to a given risk factor.
This measure is
important in quantifying the role of a specific factor in disease etiology and
in terms of the
public health impact of a risk factor. The public health relevance of this
measure lies in
estimating the proportion of cases of disease in the population that could be
prevented if the
exposure of interest were absent. AR is determined as follows:
AR=PE(RR-1)/ (PE(RR-I)+1) AR is the risk attributable to a biallelic marker
allele or a biallelic marker haplotype. PE
is the frequency of exposure to an allele or a haplotype within the population
at large; and RR is
the relative risk which, is approximated with the odds ratio when the trait
under study has a
relatively low incidence in the general population.
Association OF Biallelic Markers Of The Invention With Prostate Cancer
In the context of the present invention, an association between the purH gene
and
prostate cancer was established. Further details concerning this association
study are provided in
Example 5, results are briefly summarized below.
Two groups of independent iiidividuals were used in this association study in
accordance
with the invention: the case-control populations. The two groups corresponded
to 491 affected
individuals and 313 control individuals. The affected populations may be
subdivided in familial
cases and sporadic cases. Other subdivision can be done regarding the
diagnosis age of prostate
cancer and their familial antecedent of the disease.
In the association study described in Example 5, nuinber of biallelic marker
haplotypes
were sllown to be significantly associated with prostate cancer.
A first preferred haplotype according to the present invention (HAP I of
Figure 1 or
haplotype 3 of figure 3) comprises two biallelic markers (99-5595/380 (A29)
and 99-5596/216
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(A7)). This haplotype presented a p-value of 1.1 x10"9 and an odd-ratio of 22.
This haplotype is
significant with sporadic prostate cancer, and more significant with sporadic
cases under 65
years old. A second preferred haplotype according to the present invention
(HAP8 of Figure2 or
haplotype 4 of figure 3) comprises two biallelic markers (99-23437/347 (A20)
and 99-5596/216
(A7)). This haplotype had a p-value of 2.6x10-' and an odd ratio of 3.15 with
informative
sporadic cases. Phenotypic permutation tests confirmed the statistical
significance of these
results. These haplotypes (haplotypes 3 and 4 of figure 3) can therefore be
considered to be
highly significantly associated with prostate cancer, and more particularly
sporadic prostate
cancer.
A third preferred haplotype according to the present invention (HAP10 of
Figure 2 or
haplotype I of figure 3) comprises three biallelic markers (99-5604/376 (A30),
99-23460/199
(A17) and 99-5590/99 (A28)). This haplotype presented a p-value of 3.7xl0"5
and an odd-ratio of
2.32 for familial prostate cancer. A fourth preferred haplotype according to
the present
invention (HAP24 of Figure 2 or haplotype 2 of figure 3) comprises four
biallelic markers (99-
23452/306 (A25), 99-23440/274 (A21), 99-15798/86 (A14) and 99-5590/99 (A28)).
This
haplotype presented a p-value of 1x10-6 and an odd-ratio of 2.73 for familial
prostate cancer.
These haplotypes are significant with familial prostate cancer, and more
significant with familial
cases >=3CaP or under 65 years old. Phenotypic permutation tests confirmed the
statistical
significance of these results. Tiiese haplotypes (haplotypes I and 2 of figure
3) can therefore be
considered to be highly significantly associated with prostate cancer, and
more particularly
familial prostate cancer.
A fifth preferred haplotype according to the present invention (HAP1 of Figure
4 or
haplotype of Figure 5) comprises two markers (5-294-285 (A 10), and 99-5596-
216 (A7)) and
presented for the haplotype frequency test a p-value 2.8x10-' and an odd ratio
of 100 for the
sporadic prostate cancer. A sixth preferred haplotype according to the present
invention (HAP2
of Figure 4) comprises two biallelic markers (99-15528-333 (A 13), and 99-5596-
216 (A7)), and
presented for the haplotype frequency test a p-value of 1x10-6 and an odd-
ratio of 100 for the
sporadic prostate cancer. These haplotypes are highly significant for sporadic
prostate cancer.
The invention concerns the haplotypes associated with familial prostate cancer
comprising at least three biallelic markers selected from the group consisting
of 99-5604/376
(A30), 99-23460/199 (A17), 99-5590/99 (A28), 99-23452/306 (A25), 99-23440/274
(A21), and
99-15798/86 (A 14).
The invention concerns the haplotypes associated with sporadic prostate cancer
comprising at least two biallelic markers selected from the group consisting
of 99-5595/380
(A29), 99-5596/216 (A7) 99-23437/347 (A20). 5-294-285 (A10), and 99-15528-333
(A13).
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Preferably, the invention concerns haplotypes associated with spoaradic
prostate cancer which
comprises the biallelic 99-5596/216 (A7).
This information is extremely valuable. The knowledge of a potential genetic
predisposition to prostate cancer, even if this predisposition is not
absolute, might contribute in a
5 very significant manner to treatment efficacy of prostate cancer and to the
development of new
therapeutic and diagnostic tools.
Identification Of Biallelic Markers In Linkage Diseoluilibrium With The
Biallelic
Markers of the Invention
Once a first biallelic marker has been identified in a genomic region of
interest, the
10 practitioner of ordinary skill in the art, using the teachings of the
present invention, can easily
identify additional biallelic markers in linkage disequilibrium with this
first marker. As
mentioned before any marker in linkage disequilibrium with a first marker
associated with a trait
will be associated with the trait. Therefore, once an association has been
demonstrated between
a given biallelic marker and a trait, the discovery of additional biallelic
markers associated with
15 this trait is of great interest in order to increase the density of
biallelic markers in this particular
region. The causal gene or mutation will be found in the vicinity of the
marker or set of markers
showing the highest correlation with the trait.
Identification of additional markers in linkage disequilibrium with a given
marker
involves: (a) amplifying a genomic fragment comprising a first biallelic
marker from a plurality
20 of individuals; (b) identifying of second biallelic markers in the genomic
region harboring said
first biallelic marker; (c) conducting a linkage disequilibrium analysis
between said first biallelic
marker and second biallelic markers; and (d) selecting said second biallelic
markers as being in
linkage disequilibrium with said first marker. Subcombinations comprising
steps (b) and (c) are
also contemplated.
25 Methods to identify biallelic markers and to conduct linkage disequilibrium
analysis are
described herein and can be carried out by the skilled person without undue
experimentation.
The present invention then also concerns biallelic markers which are in
linkage disequilibrium
with the specific biallelic markers A1 to A43 and which are expected to
present similar
characteristics in terms of their respective association with a given trait.
In a preferred
30 embodiment, the invention concerns biallelic markers which are in linkage
disequilibrium with
the specific biallelic markers A29, A7, A20, A 10, and A 13, more preferably
with the biallelic
marker A7. In an otlier preferred embodiment, the invention concerns biallelic
markers which
are in linkage disequilibriuin with the specific biallelic markers A30. A17.
A28, A25, A21, and
A 14.
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Identification Of Functional Mutations
Mutations in the purH getie which are responsible for a detectable phenotype
or trait
may be identified by comparing the sequences of the purH gene from trait
positive and control
individuals. Once a positive association is confirmed with a biallelic marker
of the present
invention, the identified locus can be scanned for mutations. In a preferred
embodiment,
functional regions such as exons and splice sites, promoters and other
regulatory regions of the
purH gene are scanned for mutations. In a preferred embodiment the sequence of
the purH gene
is compared in trait positive and control individuals. Preferably, trait
positive individuals carry
the haplotype shown to be associated with the trait and trait negative
individuals do not carry the
haplotype or allele associated with the trait. The detectable trait or
phenotype may comprise a
variety of manifestations of altered purH function, including susceptibility
to prostate cancer, the
level of aggressiveness of prostate cancer tumors, an early onset of prostate
cancer, a beneficial
response to or side effects related to treatment against prostate cancer.
The mutation detection procedure is essentially similar to that used for
biallelic marker
identification. The method used to detect such mutations generally comprises
the following
steps:
- amplification of a region of the purH gene comprising a biallelic marker or
a group of
biallelic markers associated with the trait from DNA samples of trait positive
patients and trait-
negative controls;
- sequencing of the amplified region;
- comparison of DNA sequences from trait positive and control individuals;
- determination of mutations specific to trait-positive patients.
In one embodiment, said biallelic marker is selected from the group consisting
of Al to
A43, and the complements thereof. In a preferred embodiment, said biallelic
marker is selected
from the group consisting of A29, A7, A20, A10 and A 13, and the complements
thereof, more
preferably the biallelic marker A7 and the complement thereof. In a preferred
embodiment, said
biallelic marker is selected from the group consisting of A30, A17, A28. A25,
A21, and A14,
and the complements thereof. It is preferred that candidate polymorphisms be
then verified by
screening a larger population of cases and controls by means of any genotyping
procedure such
as those described herein, preferably using a microsequencing technique in an
individual test
format. Polymorphisms are considered as candidate mutations when present in
cases and
controls at frequencies compatible witli the expected association results.
Polymorphisms are
considered as candidate "trait-causing" mutations when they exhibit a
statistically significant
correlation with the detectable phenotype.
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Biallelic Markers Of The Invention In Methods Of Genetic Diagnostics
The biallelic markers of the present invention can also be used to develop
diagnostics
tests capable of identifying individuals who express a detectable trait as the
result of a specific
genotype or individuals whose genotype places them at risk of developing a
detectable trait at a
subsequent time. The trait analyzed using the present diagnostics may be any
detectable trait,
including susceptibility to prostate cancer, the level of aggressiveness of
prostate cancer tumors,
an early onset of prostate cancer, a beneficial response to or side effects
related to treatment
against prostate cancer. Such a diagnosis can be useful in the staging,
monitoring, prognosis
and/or prophylactic or curative therapy of prostate cancer.
The diagnostic techniques of the present invention may employ a variety of
methodologies to determine whether a test subject has a biallelic marker
pattern associated with
an increased risk of developing a detectable trait or whether the individual
suffers from a
detectable trait as a result of a particular mutation. including methods which
enable the analysis
of individual chromosomes for haplotyping, such as family studies, single
sperm DNA analysis
or somatic hybrids.
The present invention provides diagnostic methods to determine whether an
individual is
at risk of developing a disease or suffers from a disease resulting from a
mutation or a
polymorphism in the purH gene. The present invention also provides methods to
determine
whether an individual has a susceptibility to prostate cancer.
These methods involve obtaining a nucleic acid sample from the individual and,
determining, whether the nucleic acid sample contains at least one allele or
at least one biallelic
marker haplotype, indicative of a risk of developing the trait or indicative
that the individual
expresses the trait as a result of possessing a particular purH polymorphism
or mutation (trait-
causing allele).
Preferably, in such diagnostic methods, a nucleic acid sample is obtained from
the
individual and this sample is genotyped using methods described above in
"Methods Of
Genotyping DNA Samples For Biallelic markers. The diagnostics may be based on
a single
biallelic marker or a on group of biallelic markers.
In each of these methods, a nucleic acid sample is obtained from the test
subject and the
biallelic marker pattern of one or more of the biallelic markers A 1 to A43 is
determined.
In one embodiment, a PCR amplification is conducted on the nucleic acid sample
to
amplify regions in which polymorphisms associated with a detectable phenotype
have been
identified. The amplification products are sequenced to determine whether tiie
individual
possesses one or more purH polymorphisms associated with a detectable
phenotype. The
3 5 primers used to generate amplification products mav coniprise the primers
listed in Table I.
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Alternatively, the nucleic acid sample is subjected to microsequencing
reactions as described
above to determine whether the individual possesses one or more purH
polymorphisms
associated with a detectable phenotype resulting from a mutation or a
polymorphism in the purH
gene. The primers used in the microsequencing reactions may include the
primers listed in
Table 3. In another embodiment, the nucleic acid sample is contacted with one
or more allele
specific oligonucleotide probes which, specifically hybridize to one or more
purH alleles
associated with a detectable phenotype. The probes used in the hybridization
assay may include
the probes listed in Table 2. In another embodiment, the nucleic acid sample
is contacted with a
second purH oligonucleotide capable of producing an amplification product when
used with the
allele specific oligonucleotide in an amplification reaction. The presence of
an amplification
product in the amplification reaction indicates that the individual possesses
one or more purH
alleles associated with a detectable phenotype.
In a preferred embodiment the identity of the nucleotide present at, at least
one, biallelic
marker selected from the group consisting of A 1 to A43 and the complements
thereof, preferably
A29, A7, A20, A10 and A13, and the complements thereof, still more preferably
A7, and the -
complements thereof, is determined and the detectable trait is cancer, more
preferably prostate
cancer, more particularly sporadic prostate cancer. In a preferred embodiment
the identity of the
nucleotide present at, at least one, biallelic marker selected from the group
consisting of Al to
A43 and the complements thereof, preferably A30. A17, A28, A25, A21, and A14,
and the
complements thereof, is determined and the detectable trait is cancer, more
preferably prostate
cancer, more particularly familial prostate cancer. Diagnostic kits comprise
any of the
polynucleotides of the present invention.
These diagnostic methods are extremelv valuable as they can, in certain
circumstances,
be used to initiate preventive treatments or to allow an individual carrying a
significant
haplotype to foresee warning signs such as minor symptoms.
Diagnostics, which analyze and predict response to a drug or side effects to a
drug, may
be used to determine whether an individual should be treated with a particular
drug. For
example, if the diagnostic indicates a likelihood that an individual will
respond positively to
treatment with a particular drug, the drug may be administered to the
individual. Conversely, if
the diagnostic indicates that an individual is likely to respond negatively to
treatment with a
particular drug, an alternative course of treatment may be prescribed. A
negative response may
be defined as either the absence of an efficacious response or the presence of
toxic side effects.
Clinical drug trials represent another application for the markers of the
present
invention. One or more markers indicative of response to an agent acting
against prostate cancer
or to side effects to an agent actinQ a;ainst prostate cancer may be
identified using the methods
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described above. Thereafter, potential participants in clinical trials of such
an agent may be
screened to identify those individuals most likely to respond favorably to the
drug and exclude
those likely to experience side effects. In that way, the effectiveness of
drug treatment may be
measured in individuals who respond positively to the drug, without lowering
the measurement
as a result of the inclusion of individuals who are unlikely to respond
positively in the study and
without risking undesirable safety problems.
Treatment Of Cancer or Prostate Cancer
As the metastasis of cancer or prostate cancer can be fatal, it is important
to detect
cancer or prostate cancer susceptibility of individuals. Consequently, the
invention also
concerns a method for the treatment of cancer or prostate cancer comprising
the following steps:
- selecting an individual whose DNA comprises alleles of a biallelic marker or
of a
group of biallelic markers, preferably purH-related markers, associated with
cancer or
prostate cancer;
- following up said individual for the appearance (and optionally the
development)
of tumors in prostate or elsewhere; and
- administering an effective amount of a medicament acting against cancer or
prostate cancer to said individual at an appropriate stage of the cancer or
prostate cancer.
In one embodiment, said biallelic marker is selected from tiie group
consisting of Al to
A43, and the complements thereof. In a preferred embodiment, said biallelic
marker is selected
from the group consisting of A29, A7, A20, A 10 and A 13, and the complements
thereof, more
preferably the biallelic marker A7 and the complement thereof. In a preferred
embodiment, said
biallelic marker is selected from the group consisting of A30, A17, A28, A25,
A21, and A14,
and the complements thereof.
The prophylactic adniinistration of a treatment serves to prevent, attenuate
or inhibit the
growth of cancer cells.
Another embodiment of the present invention consists of a method for the
treatment of
cancer or prostate cancer comprising the following steps:
- selecting an individual whose DNA comprises alleles of a biallelic marker or
of a
group of biallelic markers, preferablypurH-related markers, associated with
cancer or
prostate cancer;
- administering to said individual, preferably as a preventive treatment of
cancer of
prostate cancer, an effective amount of a medicament acting against cancer or
prostate
cancer such as 4HPR.
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In one embodiment, said biallelic marker is selected from the group consisting
ofAl to
A43, and the complements thereof. In a preferred embodiment, said biallelic
marker is selected
from the group consisting of A29, A7. A20, A10 and A13, and the complements
thereof, more
preferably the biallelic marker A7 and the complement thereof. In a preferred
embodiment, said
5 biallelic marker is selected from the group consisting of A30, A 17, A28.
A25, A21, and A14,
and the complements thereof.
In a further embodiment, the present invention concerns a method for the
treatment of
cancer or prostate cancer comprising the following steps:
- selecting an individual whose DNA comprises alleles of a biallelic marker or
of a
10 group of biallelic markers, preferablypzrrH-related markers, associated
with a susceptibility
cancer or prostate cancer;
- administering to said individual, as a preventive treatment of cancer or
prostate
cancer, an effective amount of a medicament acting against cancer or prostate
cancer such
as 4HPR;
15 - following up said individual for the appearance and the development of
tumors in
prostate or elsewhere; and optionally
- administering an effective amount of a medicament acting against cancer or
prostate cancer to said individual at the appropriate stage of the cancer or
prostate cancer.
In one embodiment, said biallelic marker is selected from the group consisting
of A1 to
20 A43, and the complements thereof. In a preferred embodiment, said biallelic
marker is selected
from the group consisting of A29, A7, A20, A 10 and A 13, and the complements
thereof, more
preferably the biallelic marker A7 and the complement thereof. In a preferred
embodiment, said
biallelic marker is selected from the group consisting of A30, A 17, A28, A25,
A21, and A 14,
and the complements thereof.
25 To enlighten the choice of the appropriate beginning of the treatment of
cancer or
prostate cancer, the present invention also concerns a metliod for the
treatment of cancer or
prostate cancer comprising the following steps:
- selecting an individual suffering from a cancer or prostate cancer whose DNA
comprises alleles of a biallelic marker or of a group of biallelic markers,
preferably purH-
30 related markers, associated with the aggressiveness of cancer or prostate
cancer tumors; and
- administering an effective amount of a medicament acting against cancer or
prostate cancer to said individual.
In one embodiment, said biallelic marker is selected from the group consisting
of A1 to
A43, and the complements thereof. In a preferred embodiment, said biallelic
marker is selected
35 from the aroup consisting of A29. A7. A20. A 10 and A 13, and the
complements thereof, more
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preferably the biallelic marker A7 and the complement thereof. In a preferred
embodiment, said
biallelic marker is selected from the group consisting of A30, A17, A28, A25,
A21, and A14,
and the complements thereof.
In particular embodiments, the individual is selected by getioryping one or
more biallelic
markers of the present invention.
Recombinant Vectors
The term "vector" is used herein to designate either a circular or a linear
DNA or RNA
molecule, which is either double-stranded or single-stranded, and which
comprise at least one
polynucleotide of interest that is sought to be transferred in a cell host or
in a unicellular or
multicellular host organism.
The present invention encompasses a family of recombinant Vectors that
comprise a
regulatory polvnucleotide derived from the purH genomic sequence, or a coding
polynucleotide
from the purH genomic sequence. Consequently. the present invention further
deals with a
recombinant vector comprising either a regulatory polynucleotide comprised in
the nucleic acids
of SEQ ID No I or a polynucleotide comprising the pzrrH coding sequence or
both.
Generally, a recombinant vector of the invention may comprise any of the
polynucleotides described herein, including regulatory sequences and coding
sequences, as well
as any purH primer or probe as defined above. More particularly, the
recombinant vectors of the
present invention can comprise any of the polynucleotides described in the
"Genomic Sequences
Of tThe purHGene" section, the "purHcDNA Sequences" section, the "Coding
Regions"
section, the "Polynucleotide constructs" section, and the "Oligonucleotide
Probes And Primers"
section.
In a first preferred embodiment, a recombinant vector of the invention is used
to amplify
the inserted polynucleotide derived from a pzrrH genomic sequence of SEQ ID No
1 or apurH
cDNA, for example the cDNA of SEQ ID No 2 in a suitable cell host, this
polynucleotide being
amplified at every time that the recombinant vector replicates.
A second preferred embodiment of the recombinant vectors according to the
invention
consists of expression vectors comprising either a regulatory polynucleotide
or a coding nucleic
acid of the invention, or both. Within certain embodiments, expression vectors
are employed to
express the purH polypeptide which can be then purified and, for example be
used in ligand
screening assavs or as an immunogen in order to raise specific antibodies
directed against the
purH protein. In other embodiments, the expression vectors are used for
constructing transgenic
animals and also for gene therapy. Expression requires that appropriate
signals are provided in
the vectors, said signals including various regulatorv elements, such as
enhancers/promoters
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from both viral and mammalian sources that drive expression of the genes of
interest in host
cells. Dominant drug selection markers for establishing permanent, stable cell
clones expressing
the products are generally included in the expression vectors of the
invention, as they are
elements that link expression of the drug selection markers to expression of
the polypeptide.
More particularly, the present invention relates to expression vectors which
include
nucleic acids encoding a purH protein, preferably the purH protein of the
amino acid sequence
of SEQ ID No 3 or variants or fragments thereof, under the control of a
regulatory sequence
selected among the purH regulatory polynucleotides, or alternatively under the
control of an
exogenous regulatory sequence.
Consequently, preferred expression vectors of the invention are selected from
the group
consisting of: (a) the purH regulatory sequence comprised therein drives the
expression of a
coding polynucleotide operably linked thereto; (b) the purH coding sequence is
operably linked
to regulation sequences allowing its expression in a suitable cell host and/or
host organism.
The invention also pertains to a recombinant expression vector useful for the
expression
of the purH coding sequence, wherein said vector comprises a nucleic acid of
SEQ ID No 2.
Recombinant vectors comprising a nucleic acid containing apurH-related
bialielic
marker is also part of the invention. In a preferred embodiment, said
biallelic marker is selected
from the group consisting of A 1 to A43, and the complements thereof.
Some of the elements which can be found in the vectors of the present
invention are
described in further detail in the following sections.
1. General features of the expression vectors of the invention
A recombinant vector according to the invention comprises, but is not limited
to, a YAC
(Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a
phage, a phagemid,
a cosmid, a plasmid or even a linear DNA molecule which may consist of a
chromosomal, non-
chromosomal, semi-synthetic or synthetic DNA. Such a recombinant vector can
comprise a
transcriptional unit comprising an assembly of:
(1) a genetic element or elements having a regulatory role in gene expression,
for
example promoters or enhancers. Enhancers are cis-acting elements of DNA,
usually from
about 10 to 300 bp in length that act on the promoter to increase the
transcription.
(2) a structural or coding sequence which is transcribed into mRNA and
eventually
translated into a polypeptide, said structural or coding sequence being
operably linked to the
regulatory elements described in (1); and
(3) appropriate transcription initiation and termination sequences. Structural
units
intended for use in yeast or eukaryotic expression systeins preferably include
a leader sequence
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enabling extracellular secretion of translated protein by a host cell.
Alternatively, when a
recombinant protein is expressed without a leader or transport sequence, it
may include a N-
terminal residue. This residue inay or may not be subsequently cleaved from
the expressed
recombinant protein to provide a final product.
Generally, recombinant expression vectors will include origins of replication,
selectable
markers permitting transformation of the host cell, and a promoter derived
from a highly
expressed gene to direct transcription of a downstream structural sequence.
The heterologous
structural sequence is assembled in appropriate phase with translation
initiation and termination
sequences, and preferably a leader sequence capable of directing secretion of
the translated
protein into the periplasmic space or the extracellular medium. In a specific
embodiment
wherein the vector is adapted for transfecting and expressing desired
sequences in mammalian
host cells, preferred vectors will comprise an origin of replication in the
desired host, a suitable
promoter and enhancer, and also any necessary ribosome binding sites,
polyadenylation site,
splice donor and acceptor sites, transcriptional termination sequences, and 5'-
flanking non-
transcribed sequences. DNA sequences derived from the SV40 viral genome, for
example SV40
origin, early promoter, enhancer, splice and polyadenylation sites may be used
to provide the
required non-transcribed genetic elements.
The in vivo expression of a purH polypeptide of SEQ ID No 3 or fragments or
variants
thereof may be useful in order to correct a genetic defect related to the
expression of the native
gene in a host organism or to the production of a biologically inactive purH
protein.
Consequently, the present invention also deals with recombinant expression
vectors
mainly designed for the in vivo production of the purH polypeptide of SEQ ID
No 3 or
fragments or variants thereof by the introduction of the appropriate genetic
material in the
organism of the patient to be treated. This genetic material may be introduced
in vitro in a cell
that has been previously extracted from the organism, the modified cell being
subsequently
reintroduced in the said organism, directly in vivo into the appropriate
tissue.
2. Regulatory Elements
Promoters
The suitable promoter regions used in the expression vectors according to the
present
invention are chosen taking into account the cell host in which the
heterologous gene has to be
expressed. The particular promoter employed to control the expression of a
nucleic acid
sequence of interest is not believed to be important, so long as it is capable
of directing the
expression of the nucleic acid in the targeted cell. Thus, where a liuman cell
is targeted, it is
preferable to position the nucleic acid coding re~ion adjacent to and under
the control of a
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promoter that is capable of being expressed in a human cell, such as, for
example, a human or a
viral promoter.
A suitable promoter may be heterologous with respect to the nucleic acid for
which it
controls the expression or alternatively can be endogenous to the native
polynucleotide
containing the coding sequence to be expressed. Additionally, the promoter is
generally
heterologous with respect to the recombinant vector sequences within which the
construct
promoter/coding sequence has been inserted.
Promoter regions can be selected from any desired gene using, for example, CAT
(chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7
vectors.
Preferred bacterial promoters are the LacI, LacZ, the T3 or T7 bacteriophage
RNA
polymerase promoters, the gpt, lambda PR, PL and trp promoters (EP 0036776),
the polyhedrin
promoter, or the p10 protein promoter from baculovirus (Kit Novagen) (Smith et
al., 1983;
O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.
Eukaryotic promoters include CMV immediate early. HSV thymidine kinase, early
and
late SV40, LTRs from retrovirus, and mouse metallothionine-L. Selection of a
convenient
vector and promoter is well within the level of ordinary skill in the art.
The choice of a promoter is well within the ability of a person skilled in the
fteld of
genetic engineering. For example, one may refer to the book of Sambrook et
al.(1989) or also to
the procedures described by Fuller et al.(1996).
Other regulatory elements
Where a cDNA insert is employed, one will typically desire to include a
polyadenylation
signal to effect proper polyadenylation of the gene transcript. The nature of
the polyadenylation
signal is not believed to be crucial to the successful practice of the
invention, and any such
sequence may be employed such as human growth hormone and SV40 polyadenylation
signals.
Also contemplated as an element of the expression cassette is a terminator.
These elements can
serve to enhance message levels and to minimize read through from the cassette
into other
sequences.
The vector containing the appropriate DNA sequence as described above, more
preferably purH gene regulatory polynucleotide. a polynucleotide encoding the
palrH
polypeptide selected from the aroup consisting of SEQ ID No I or a fragment or
a variant
thereof and SEQ ID No 2, or both of them, can be utilized to transform an
appropriate host to
allow the expression of the desired polypeptide or polynucleotide.
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3. Selectable Markers
Such markers would confer an identifiable change to the cell permitting easy
identification of cells containing the expression construct. The selectable
marker genes for
selection of transformed host cells are preferably dihvdrofolate reductase or
neomycin resistance
5 for eukaryotic cell culture, TRP1 for S. cerevisiae or tetracycline,
rifampicin or ampicillin
resistance in E. coli, or levan saccharase for mycobacteria, this latter
marker being a negative
selection marker.
4. Preferred Vectors.
Bacterial vectors
10 As a representative but non-limiting example, useful expression vectors for
bacterial use
can comprise a selectable marker and a bacterial origin of replication derived
from commercially
available plasmids comprising genetic elements of pBR322 (ATCC 37017). Such
commercial
vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and GEM1
(Promega
Biotec, Madison, WI, USA).
15 Large numbers of other suitable vectors are known to those of skill in the
art, and
commerciallv available, such as the following bacterial vectors: pQE70, pQE60,
pQE-9
(Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A,
pNH16A, pNH18A,
pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia);
pWLNEO, pSV2CAT, pOG44, pXTI, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL
20 (Pharmacia); pQE-30 (QlAexpress).
Bacteriopha_ge vectors
The P 1 bacteriophage vector may contain large inserts ranging from about 80
to about
100 kb.
The construction of P 1 bacteriophage vectors such as p 158 or p 158/neo8 are
notably
25 described by Sternberg (1992, 1994). Recombinant P 1 clones comprising purH
nucleotide
sequences mav be designed for inserting large polvnucleotides of more than 40
kb (Linton et al.,
1993). To generate P1 DNA for transgenic experiments, a preferred protocol is
the protocol
described by McCormick et al.(1994). Briefly, E. coli (preferably strain
NS3529) harboring the
P1 plasmid are grown overnight in a suitable broth medium containing 25 g/ml
of kanamycin.
30 The P1 DNA is prepared from the E. coli by alkaline lysis using the Qiagen
Plasmid Maxi kit
(Qiagen, Chatsworth, CA, USA), according to the manufacturer's instructions.
The P1 DNA is
purified from the bacterial lvsate on two Qiagen-tip 500 columns, using the
washing and elution
buffers contained in the kit. A phenol/chloroform extraction is tlien
performed before
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precipitating the DNA with 70% ethanol. After solubilizing the DNA in TE (10
mM Tris-HCI,
pH 7.4, 1 mM EDTA), the concentration of the DNA is assessed by
spectrophotometry.
When the goal is to express a P 1 clone comprising purH nucleotide sequences
in a
transgenic animal, typically in transgenic mice, it is desirable to remove
vector sequences from
the PI DNA fragment, for example by cleaving the PI DNA at rare-cutting sites
within the P1
polylinker (SfiI. Notl or Sall). The P I insert is then purified from vector
sequences on a pulsed-
field agarose gel. using methods similar using methods similar to those
originally reported for
the isolation of DNA from YACs (Schedl et al., 1993a; Peterson et al., 1993).
At this stage, the
resulting purified insert DNA can be concentrated, if necessary, on a
Millipore Ultrafree-MC
Filter Unit (Millipore, Bedford, MA, USA - 30,000 molecular weight limit) and
then dialyzed
against microinjection buffer (10 mM Tris-HCI, pH 7.4; 250 M EDTA) containing
100 mM
NaCI, 30 M spermine, 70 M spermidine on a microdyalisis membrane (type VS,
0.025 M
from Millipore). The intactness of the purified P1 DNA insert is assessed by
electrophoresis on
1% agarose (Sea Kem GTG; FMC Bio-products) pulse-field gel and staining with
ethidium
bromide.
Baculovirus vectors
A suitable vector for the expression of the purH polypeptide of SEQ ID No 3 or
fragments or variants thereof is a baculovirus vector that can be propagated
in insect cells and in
insect cell lines. A specific suitable host vector system is the pVL1392/1393
baculovirus
transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC
N CRL 1711)
which is derived from Spodoptera frugiperda.
Other suitable vectors for the expression of the purH polypeptide of SEQ ID No
3 or
fragments or N-ariants thereof in a baculovirus expression system include
those described by Chai
et al.(1993), Viasak et al.(1983) and Lenhard et al.(1996).
Viral vectors
In one specific embodiment, the vector is derived from an adenovirus.
Preferred
adenovirus vectors according to the invention are those described by Feldman
and Steg (1996) or
Ohno et al.(1994). Another preferred recombinant adenovirus according to this
specific
embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2
or Ad 5) or an
adenovirus of animal origin ( French patent application N FR-93.05954).
Retrovirus vectors and adeno-associated virus vectors are generally understood
to be the
recombinant gene delivery systems of choice for the transfer of exogenous
polynucleotides in
vivo , particularly to mammals, including humans. These vectors provide
efficient delivery of
genes into cells. and the transferred nucleic acids are stably integrated into
the chromosomal
DNA of the host.
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Particularly preferred retroviruses for the preparation or construction of
retroviral in
vitro or in vitro gene delivery vehicles of the present invention include
retroviruses selected from
the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus,
Reticuloendotheliosis virus and Rous Sarcoma virus. Particularly preferred
Murine Leukemia
Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999),
Friend (ATCC
No VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney
Murine
Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298). Particularly
preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-
657, VR-
726, VR-659 and VR-728). Other preferred retroviral vectors are those
described in Roth et
al.(1996), PCT Application No WO 93/25234, PCT Application No WO 94/ 06920,
Roux et al.,
1989, Julan et al., 1992 and Neda et al., 1991, the disclosures of which are
incorporated by
reference herein in their entirety.
Yet another viral vector system that is contemplated by the invention consists
in the
adeno-associated virus (AAV). The adeno-associated virus is a naturally
occurring defective
virus that requires another virus, such as an adenovirus or a herpes virus, as
a helper virus for
efficient replication and a productive life cycle (Muzyczka et al., 1992). It
is also one of the few
viruses that may integrate its DNA into non-dividing cells, and exhibits a
high frequency of
stable integration (Flotte et al., 1992; Samulski et al., 1989; McLaughlin et
al., 1989). One
advantageous feature of AAV derives from its reduced efficacy for transducing
primary cells
relative to transformed cells.
BAC vectors
The bacterial artificial chromosome (BAC) cloning system (Shizuya et al..
1992) has
been developed to stably maintain large fragments of genomic DNA (100-300 kb)
in E. coli. A
preferred BAC vector consists of pBeIoBAC 1 1 vector that has been described
by Kim et
al.(1996). BAC libraries are prepared with this vector using size-selected
genomic DNA that has
been partially digested using enzymes that permit ligation into either the
Barn HI or HindIIl sites
in the vector. Flanking these cloning sites are T7 and SP6 RNA polymerase
transcription
initiation sites that can be used to generate end probes by either RNA
transcription or PCR
methods. After the construction of a BAC library in E. coli, BAC DNA is
purified from the
host cell as a supercoiled circle. Converting these circular molecules into a
linear form precedes
both size determination and introduction of the BACs into recipient cells. The
cloning site is
flanked by two Not I sites, permitting cloned segments to be excised from the
vector by Not I
digestion. Alternatively, the DNA insert contained in the pBeIoBAC I I vector
may be linearized
by treatment of the BAC vector with the commercially available enzyme lambda
terminase that
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leads to the cleavage at the unique cosN site, but this cleavage method
results in a full length
BAC clone containing both the insert DNA and the BAC sequences.
5. Delivery Of The Recombinant Vectors
In order to effect expression of the polynucleotides and polynucleotide
constructs of the
invention, these constructs must be delivered into a cell. This delivery may
be accomplished in
vitro, as in laboratory procedures for transforming cell lines, or in vivo or
ex vivo, as in the
treatment of certain diseases states.
One mechanism is viral infection where the expression construct is
encapsulated in an
infectious viral particle.
Several non-viral methods for the transfer of polynucleotides into cultured
mammalian
cells are also contemplated by the present invention, and include, without
being limited to,
calcium phosphate precipitation (Graham et al., 1973; Chen et al., 1987;),
DEAE-dextran
(Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),
direct microinjection
(Harland et al., 1985), DNA-loaded liposomes (Nicolau et al., 1982; Fralev et
al., 1979), and
receptor-mediate transfection (Wu and Wu, 1987; 1988). Some of these
techniques may be
successfully adapted for in vivo or ex vivo use.
Once the expression polynucleotide has been delivered into the cell, it may be
stably
integrated into the genome of the recipient cell. This integration may be in
the cognate location
and orientation via homologous recombination (gene replacement) or it may be
integrated in a
random, non specific location (gene augmentation). In yet further embodiments,
the nucleic acid
may be stably maintained in the cell as a separate, episomal segment of DNA.
Such nucleic acid
segments or "episomes" encode sequences sufficient to permit maintenance and
replication
independent of or in synchronization with the host cell cycle.
One specific embodiment for a method for delivering a protein or peptide to
the interior
of a cell of a vertebrate in vivo comprises the step of introducing a
preparation comprising a
physiologically acceptable carrier and a naked polvnucleotide operatively
coding for the
polypeptide of interest into the interstitial space of a tissue comprising the
cell, whereby the
naked polynucleotide is taken up into the interior of the cell and has a
physiological effect. This
is particularly applicable for transfer in vitro but it may be applied to in
vivo as well.
Compositions for use in vitro and in vivo comprising a"naked" polynucleotide
are
described in PCT application N WO 90/1 1092 (Vical Inc.) and also in PCT
application No. WO
95/11307 (Institut Pasteur, INSERM, Universite d'Ottawa) as well as in the
articles of Tacson et
al.(1996) and of Huygen et al.(1996)., the disclosures of which are
incorporated by reference
herein in their entirety
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In still another embodiment of the invention, the transfer of a naked
polynucleotide of
the invention, including a polynucleotide construct of the invention, into
cells may be proceeded
with a particle bombardment (biolistic), said particles being DNA-coated
microprojectiles
accelerated to a high velocity allowing them to pierce cell membranes and
enter cells without
killing them, such as described by Klein et al.(1987).
In a further embodiment, the polynucleotide of the invention may be entrapped
in a
liposome (Ghosh and Bacchawat, 1991; Wong et al., 1980; Nicolau et al., 1987)
In a specific embodiment, the invention provides a composition for the in vivo
production of the purH protein or polypeptide described herein. It comprises a
naked
polynucleotide operatively coding for this polypeptide, in solution in a
physiologically
acceptable carrier, and suitable for introduction into a tissue to cause cells
of the tissue to
express the said protein or polypeptide.
The amount of vector to be injected to the desired host organism varies
according to the
site of injection. As an indicative dose, it will be injected between 0,1 and
100 g of the vector
in an animal body, preferably a mammal body, for example a mouse body.
In another embodiment of the vector according to the invention, it may be
introduced in
vitro in a host cell, preferably in a host cell previously harvested from the
animal to be treated
and more preferably a somatic cell such as a muscle cell. In a subsequent
step, the cell that has
been transformed with the vector coding for the desired purH polypeptide or
the desired
fragment thereof is reintroduced into the animal body in order to deliver the
recombinant protein
within the bodv either locally or systemicallv.
Cell Hosts
Another object of the invention consists of a host cell that has been
transformed or
transfected with one of the polynucleotides described herein, and in
particular a polynucleotide
either comprising apurH regulatory polynucleotide or the coding sequence of
the purH
polypeptide selected from the group consisting of SEQ ID No I or a fragment or
a variant
thereof and SEQ ID No 2. Also included are host cells that are transformed
(prokaryotic cells)
or that are transfected (eukaryotic cells) with a recombinant vector such as
one of those
described above.
Generally, a recombinant host cell of the invention comprises any one of the
polynucleotides or the recombinant vectors described herein. More
particularly, the cell hosts of
the present invention can comprise any of the polynucleotides described in the
"Genomic
Sequences Of tThe purH Gene" section, the "purH cDNA Sequences" section, the
"Coding
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Regions" section, the "Polynucleotide constructs" section, and the
"Oligonucleotide Probes And
Primers" section.
A further recombinant cell host according to the invention comprises a
polynucleotide
containing a biallelie marker selected from the group consisting of Al to A43,
and the
complements thereof.
Preferred host cells used as recipients for the expression vectors of the
invention are the
following:
a) Prokaryotic host cells: Escherichia coli strains (I.E.DH5-a strain),
Bacillus subtilis,
Salmonella tvphinzurium, and strains from species like Pseudomonas,
Streptomyces and
Staphylococcus.
b) Eukaryotic host cells: HeLa cells (ATCC N CCL2; N CCL2.1; N CCL2.2), Cv I
cells (ATCC N CCL70), COS cells (ATCC N CRL1650; N CRL1651), Sf-9 cells (ATCC
N CRL1711), C127 cells (ATCC N CRL-1804), 3T3 (ATCC N CRL-6361), CHO (ATCC N
CCL-61), human kidney 293. (ATCC N 45504; N CRL-1573) and BHK (ECACC N
84100501; N 841 11301).
c) Other mammalian host cells.
The purH gene expression in mammalian, and typically human, cells may be
rendered
defective, or alternatively it may be proceeded with the insertion of a purH
genomic or cDNA
sequence with the replacement of the purH gene counterpart in the genome of an
animal cell by
a purH polynucleotide according to the invention. These genetic alterations
may be generated
by homologous recombination events using specific DNA constructs that have
been previously
described.
One kind of cell hosts that may be used are mammal zygotes, such as murine
zygotes.
For example, murine zygotes may undergo microinjection with a purified DNA
molecule of
interest, for example a purified DNA molecule that has previously been
adjusted to a
concentration range from I ng/ml -for BAC inserts- 3 ng/ l -for Pl
bacteriophage inserts- in 10
mM Tris-HCI. pH 7.4, 250 M EDTA containing 100 mM NaCI, 30 M spermine, and70
M
spermidine. When the DNA to be microinjected has a large size, polyamines and
high salt
concentrations can be used in order to avoid mechanical breakage of this DNA,
as described by
Schedl et al (1993b).
Anvone of the polvnucleotides of the invention, including the DNA constructs
described
herein, may be introduced in an embryonic stem (ES) cell line, preferably a
mouse ES cell line.
ES cell lines are derived from pluripotent, uncommitted cells of the inner
cell mass of pre-
implantation blastocysts. Preferred ES cell lines are the following: ES-
E14TG2a (ATCC n
CRL-1821), ES-D3 (ATCC n CRL1934 and n CRL-1 1632). YS001 (ATCC n CRL-
l1776),
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36.5 (ATCC n CRL-11116). To maintain ES cells in an uncommitted state, they
are cultured in
the presence of growth inhibited feeder cells which provide the appropriate
signals to preserve
this embryonic phenotype and serve as a matrix for ES cell adlierence.
Preferred feeder cells
consist of primary embryonic fibroblasts that are established from tissue of
day 13- day 14
embryos of virtually any mouse strain, that are maintained in culture, such as
described by
Abbondanzo et al.(1993) and are inhibited in growth by irradiation, such as
described by
Robertson (1987), or by the presence of an inhibitory concentration of LIF,
such as described by
Pease and Williams (1990).
The constructs in the host cells can be used in a conventional manner to
produce the
gene product encoded by the recombinant sequence.
Following transformation of a suitable host and growth of the host to an
appropriate cell
density, the selected promoter is induced by appropriate means, such as
temperature shift or
chemical induction, and cells are cultivated for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or
chemical means,
and the resulting crude extract retained for further purification.
Microbial cells einployed in the expression of proteins can be disrupted by
any
convenient method, including freeze-thaw cycling, sonication, mechanical
disruption, or use of
cell lysing agents. Such methods are well known by the skill artisan.
Trans2enic Animals
The terms "transgenic animals" or "host animals" are used herein designate
animals that
have their genome genetically and artificially manipulated so as to include
one of the nucleic
acids accordin(y to the invention. Preferred animals are non-human mammals and
include those
belonging to a genus selected from A1zts (e.g. mice), Rattus (e.g. rats) and
Oiyctogalus (e.g.
rabbits) which have their genome artificially and genetically altered by the
insertion of a nucleic
acid accordinQ to the invention. In one embodiment, the invention encompasses
non-human host
mammals and animals comprising a recombinant vector of the invention or a purH
gene
disrupted by homologous recombination with a knock out vector.
The transgenic animals of the invention all include within a pluralitv of
their cells a
cloned recombinant or synthetic DNA sequence, more specifically one of the
purified or isolated
nucleic acids comprising a patrH coding sequence, a purH regulatory
polvnucleotide or a DNA
sequence encoding an antisense polynucleotide such as described in the present
specification.
Generally. a transgenic animal according the present invention comprises any
one of the
polynucleotides, the recombinant vectors and the cell hosts described in the
present invention.
More particularly, the transgenic animals of the present invention can
comprise anv of the
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polynucleotides described in the "Genomic Sequences Of tThe purHGene" section,
the "purH
cDNA Sequences" section, the "Coding Regions" section, the "Polvnucleotide
constructs"
section, the "Oligonucleotide Probes And Primers" section, the "Recombinant
Vectors" section
and the "Cell Hosts" section.
A further transgenic animals according to the invention contains in their
somatic cells
and/or in their germ line cells a polynucleotide comprising a biallelic marker
selected from the
group consisting of AI to A43, and the complements thereof.
In a first preferred embodiment, these transgenic animals may be good
experimental
models in order to study the diverse pathologies related to cell
differentiation, in particular
concerning the transgenic animals within the genome of which has been inserted
one or several
copies of a polynucleotide encoding a native purH protein, or alternatively a
mutant purH
protein.
In a second preferred embodiment, these transgenic animals may express a
desired
polypeptide of interest under the control of the regulatory polynucleotides of
the purH gene,
leading to good yields in the synthesis of this protein of interest, and
eventually a tissue specific
expression of this protein of interest.
The design of the transgenic animals of the invention may be made according to
the
conventional techniques well known from the one skilled in the art. For more
details regarding
the production of transgenic animals, and specifically transgenic mice, it may
be referred to US
Patents Nos 4,873,191, issued Oct. 10, 1989; 5,464,764 issued Nov 7, 1995; and
5,789,215,
issued Aug 4, 1998; these documents being herein incorporated by reference to
disclose methods
producing transgenic mice.
Transgenic animals of the present invention are produced by the application of
procedures which result in an animal with a genome that has incorporated
exogenous genetic
material. The procedure involves obtaining the genetic material, or a portion
thereof, which
encodes either a purH coding sequence, a purH regulatory polynucleotide or a
DNA sequence
encoding a pzrrH antisense polynucleotide such as described in the present
specification.
A recoinbinant polvnucleotide of the invention is inserted into an embryonic
or ES stem
cell line. The insertion is preferably made using electroporation, such as
described by Thomas et
al.(1987). The cells subjected to electroporation are screened (e.g. by
selection via selectable
markers, by PCR or by Southern blot analysis) to find positive cells which
have integrated the
exogenous recombinant polynucleotide into their genome, preferably via an
homologous
recombination event. An illustrative positive-negative selection procedure
that may be used
according to the invention is described by Mansour et al.(1988).
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Then, the positive cells are isolated, cloned and injected into 3.5 days old
blastocysts
from mice, such as described by Bradley (1987). The blastocysts are then
inserted into a female
host animal and allowed to grow to term.
Alternatively, the positive ES cells are brought into contact with embryos at
the 2.5 days
old 8-16 cell stage (morulae) such as described bv Wood et al.(1993) or by
Nagy et al.(1993),
the ES cells being internalized to colonize extensively the blastocyst
including the cells which
will give rise to the germ line.
The offspring of the female host are tested to determine which animais are
transgenic
e.g. include the inserted exogenous DNA sequence and which are wild-type.
Thus, the present invention also concerns a transgenic animal containing a
nucleic acid,
a recombinant expression vector or a recombinant host cell according to the
invention.
Recombinant Cell Lines Derived From The Transgenic Animals Of The Invention.
A further object of the invention consists of recombinant host cells obtained
from a
transgenic animal described herein. In one embodiment the invention
encompasses cells derived
from non-human host mammals and animals comprising a recombinant vector of the
invention
or a purH gene disrupted by homologous recombination with a knock out vector.
Recombinant cell lines may be established in vitro from cells obtained from
any tissue of
a transgenic animal according to the invention, for example by transfection of
primary cell
cultures with vectors expressing onc-genes such as SV40 large T antigen, as
described by Chou
(1989) and Shay et al.(1991).
Method For Screeninil Substances Interactin2 With The Regulatorv Sequences Of
The pcrrH Gene.
The present invention also concerns a method for screening substances or
molecules that
are able to interact with the regulatory sequences of the pz+rH gene, such as
for example
promoter or enhancer sequences.
Nucleic acids encoding proteins which are able to interact with the regulatory
sequences
of the purH gene, more particularlv a nucleotide sequence selected from the
group consisting of
the polynucleotides of the 5' and 3' re-ulatorv region or a fragment or
variant thereof, and
preferably a variant comprising one of the biallelic niarkers of the
invention, may be identified
by using a one-hybrid system, such as that described in the booklet enclosed
in the Matchmaker
One-Hybrid System kit from Clontech (Catalog Ref. n K 1603-1), the technical
teachings of
which are herein incorporated bv reference. Briefly, the target nucleotide
sequence is cloned
upstream of a selectable reporter sequence and the resulting DNA construct is
integrated in the
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yeast genome (Saccharomyces cerevisiae). The yeast cells containing the
reporter sequence in
their genome are then transformed with a library consisting of fusion
molecules between cDNAs
encoding candidate proteins for binding onto the regulatory sequences of the
purH gene and
sequences encoding the activator domain of a yeast transcription factor such
as GAL4. The
recombinant yeast cells are plated in a culture broth for selecting cells
expressing the reporter
sequence. The recombinant yeast cells thus selected contain a fusion protein
that is able to bind
onto the target regulatory sequence of the purH gene. Then, the cDNAs encoding
the fusion
proteins are sequenced and may be cloned into expression or transcription
vectors in vitro. The
binding of the encoded polypeptides to the target regulatory sequences of the
purH gene may be
confirmed by techniques familiar to the one skilled in the art, such as gel
retardation assays or
DNAse protection assays.
Gel retardation assays may also be performed independently in order to screen
candidate
molecules that are able to interact with the regulatory sequences of the purH
gene, such as
described by Fried and Crothers (1981), Gamer and Revzin (1981) and Dent and
Latchman
(1993), the teachings of these publications being herein incorporated by
reference. These
techniques are based on the principle according to which a DNA fragment which
is bound to a
protein migrates slower than the same unbound DNA fragment. Briefly, the
target nucleotide
sequence is labeled. Then the labeled target nucleotide sequence is brought
into contact with
either a total nuclear extract from cells containing transcription factors, or
with different
candidate molecules to be tested. The interaction between the target
regulatory sequence of the
purH gene and the candidate molecule or the transcription factor is detected
after gel or capillary
electrophoresis through a retardation in the migration.
Method For Screenin$! Li2ands That Modulate The Expression Of The purH Gene.
Another subject of the present invention is a method for screening molecules
that
modulate the expression of the purH protein. Such a screening method comprises
the steps of:
a) cultivating a prokaryotic or an eukaryotic cell that has been transfected
with a
nucleotide sequence encoding the purH protein or a variant or a fragment
thereof, placed under
the control of its own promoter;
b) bringing into contact the cultivated cell with a molecule to be tested;
c) quantifving the expression of the purH protein or a variant or a fragment
thereof.
In an embodiment, the nucleotide sequence encoding the purH protein or a
variant or a
fragment thereof comprises an allele of at least one of the biallelic markers
AI to A17. A34 and
A35, and the complements thereof.
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Using DNA recombination techniques well known by the one skill in the art, the
purH
protein encoding DNA sequence is inserted into an expression vector,
downstream from its
promoter sequence. As an illustrative example, the promoter sequence of the
purH gene is
contained in the nucleic acid of the 5' regulatory region.
The quantification of the expression of the purH protein may be realized
either at the
mRNA level or at the protein level. In the latter case, polyclonal or
monoclonal antibodies may
be used to quantify the amounts of the purH protein that have been produced,
for example in an
ELISA or a RIA assay.
In a preferred embodiment, the quantification of the purH mRNA is realized by
a
quantitative PCR amplification of the cDNA obtained by a reverse transcription
of the total
mRNA of the cultivated purH -transfected host cell, using a pair of primers
specific for purH.
The present invention also concerns a method for screening substances or
molecules that
are able to increase, or in contrast to decrease. the level of expression of
the pzrrH gene. Such a
method may allow the one skilled in the art to select substances exerting a
regulating effect on
the expression level of the purH gene and which may be useful as active
ingredients included in
pharmaceutical compositions for treating patients suffering from prostate
cancer.
Thus, is also part of the present invention a method for screening of a
candidate
substance or molecule that modulated the expression of the pzrrH gene, this
method comprises
the following steps:
- providing a recombinant cell host containing a nucleic acid, wherein said
nucleic acid
comprises a nucleotide sequence of the 5' regulatory region or a biologically
active fragment or
variant thereof located upstream a polynucleotide encoding a detectable
protein;
- obtaining a candidate substance; and
- determining the ability of the candidate substance to modulate the
expression levels of
the polynucleotide encoding the detectable protein.
In a further embodiment, the nucleic acid comprising the nucleotide sequence
of the 5'
regulatory region or a biologically active fragment or variant thereof also
includes a 5'UTR
region of the purH cDNA of SEQ ID No 2, or one of its biologically active
fragments or variants
thereof.
Amona the preferred polynucleotides encoding a detectable protein, there may
be cited
polynucleotides encoding beta galactosidase, green fluorescent protein (GFP)
and
chloramphenicol acetyl transferase (CAT).
The invention also pertains to kits useful for performin(i the herein
described screening
method. Preferably, such kits comprise a recombinant vector that allows the
expression of a
nucleotide sequence of the 5' regulatorv region or a biologicallv active
fragment or variant
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thereof located upstream and operably linked to a polynucleotide encoding a
detectable protein
or the purH protein or a fragment or a variant thereof.
In another embodiment of a method for the screening of a candidate substance
or
molecule that modulates the expression of the purH gene, wherein said method
comprises the
following steps:
a) providing a recombinant host cell containing a nucleic acid, wherein said
nucleic acid
comprises a 5'UTR sequence of the purH cDNA of SEQ ID No 2, or one of its
biologically
active fragments or variants, the 5'UTR sequence or its biologically active
fragment or variant
being operably linked to a polynucleotide encoding a detectable protein;
b) obtaining a candidate substance; and
c) determining the ability of the candidate substance to modulate the
expression levels of
the polynucleotide encoding the detectable protein.
In a specific embodiment of the above screening method, the nucleic acid that
comprises
a nucleotide sequence selected from the group consisting of the 5'UTR sequence
of the purH
cDNA of SEQ ID No 2 or one of its biologically active fragments or variants,
includes a
promoter sequence which is endogenous with respect to the purH 5'UTR sequence.
In another specific embodiment of the above screening method, the nucleic acid
that
comprises a nucleotide sequence selected from the group consisting of the
5'UTR sequence of
the purH cDNA of SEQ ID No 2 or one of its biologically active fragments or
variants, includes
a promoter sequence which is exogenous with respect to the purH 5'UTR sequence
defined
therein.
In a further preferred embodiment, the nucleic acid comprising the 5'-UTR
sequence of
the pzrrH cDNA or SEQ ID No 2 or the biologically active fragments thereof
includes a biallelic
marker selected from the group consisting of A 1 to A17, A34 and A35 or the
complements
thereof.
The invention further deals with a kit for the screening of a candidate
substance
modulating the expression of the purH gene, wherein said kit comprises a
recombinant vector
that comprises a nucleic acid including a 5'UTR sequence of the purH cDNA of
SEQ ID No 2,
or one of their biologically active fragments or variants, the 5'UTR sequence
or its biologically
active fragment or variant being operably linked to a polynucleotide encoding
a detectable
protein.
For the design of suitable recombinant vectors useful for performing the
screening
methods described above, it will be referred to the section of the present
specification wherein
the preferred recombinant vectors of the invention are detailed.
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Expression levels and patterns of purH may be analyzed by solution
hybridization with
long probes as described in International Patent Application No. WO 97/05277,
the entire
contents of which are incorporated lierein by reference. Briefly, the purH
cDNA or the purH
genomic DNA described above, or fragments thereof, is inserted at a cloning
site immediately
downstream of a bacteriophage (T3, T7 or SP6) RNA polymerase promoter to
produce antisense
RNA. Preferably, the purH insert comprises at least 100 or more consecutive
nucleotides of the
genomic DNA sequence or the cDNA sequences. The plasmid is linearized and
transcribed in
the presence of ribonucleotides comprising modified ribonucleotides (i.e.
biotin-UTP and DIG-
UTP). An excess of this doubly labeled RNA is hybridized in solution with mRNA
isolated
from cells or tissues of interest. The hybridization is performed under
standard stringent
conditions (40-50 C for 16 hours in an 80% formamide, 0. 4 M NaCI buffer, pH 7-
8). The
unhybridized probe is removed by digestion with ribonucleases specific for
single-stranded RNA
(i.e. RNases CL3, T1, Phy M, U2 or A). The presence of the biotin-UTP
modification enables
capture of the hybrid on a microtitration plate coated with streptavidin. The
presence of the DIG
modification enables the hybrid to be detected and quantified by ELISA using
an anti-DIG
antibody coupled to alkaline phosphatase.
Quantitative analysis of purH gene expression may also be performed using
arrays. As
used herein, the term array means a one dimensional, two dimensional, or
multidimensional
arrangement of a plurality of nucleic acids of sufficient length to permit
specific detection of
expression of mRNAs capable of hybridizing thereto. For example, the arrays
may contain a
plurality of nucleic acids derived from genes whose expression levels are to
be assessed. The
arrays may include the purH genomic DNA, the purH cDNA sequences or the
sequences
complementary thereto or fragments thereof, particularly those comprising at
least one of the
biallelic markers according the present invention, preferably at least one of
the biallelic markers
A] to A43. Preferably, the fragments are at least 15 nucleotides in length. In
other
embodiments, the fragments are at least 25 nucleotides in length. In some
embodiments, the
fragments are at least 50 nucleotides in length. More preferably, the
fragments are at least 100
nucleotides in length. In another preferred embodiment, the fragments are more
than 100
nucleotides in length. In some embodiments the fragments may be more than 500
nucleotides in
length.
For example, quantitative analysis of purH gene expression may be performed
with a
complementary DNA microarray as described by Schena et al.(1995 and 1996).
Full length
purHcDNAs or fragments thereof are amplified by PCR and arrayed from a 96-well
microtiter
plate onto silylated microscope slides using high-speed robotics. Printed
arrays are incubated in
a humid chamber to allow rehvdration of the arrav elements and rinsed, once in
0. 2% SDS for 1
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min, twice in water for I min and once for 5 min in sodium borohydride
solution. The arrays are
submerged in water for 2 min at 95 C, transferred into 0. 2% SDS for I min,
rinsed twice with
water, air dried and stored in the dark at 25 C.
Cell or tissue mRNA is isolated or commercially obtained and probes are
prepared by a
single round of reverse transcription. Probes are hybridized to 1 cm'
microarrays under a 14 x
14 mm glass coverslip for 6-12 hours at 60 C. Arrays are washed for 5 min at
25 C in low
stringency wash buffer (1 x SSC/0. 2% SDS), then for 10 min at room
temperature in high
stringency wash buffer (0. 1 x SSC/0. 2% SDS). Arrays are scanned in 0. 1 x
SSC using a
fluorescence laser scanning device fitted with a custom filter set. Accurate
differential
expression measurements are obtained by taking the average of the ratios of
two independent
hybridizations.
Quantitative analysis of purH gene expression may also be performed with full
length
pzrrH cDNAs or fragments thereof in complementary DNA arrays as described by
Pietu et
al.(1996). The full length purH cDNA or fragments thereof is PCR amplified and
spotted on
membranes. Then, mRNAs originating from various tissues or cells are labeled
with radioactive
nucleotides. After hybridization and washing in controlled conditions, the
hybridized mRNAs
are detected by phospho-imaging or autoradiography. Duplicate experiments are
performed and
a quantitative analysis of differentially expressed mRNAs is then performed.
Alternatively, expression analysis using the purH genomic DNA, the purH cDNA,
or
fragments thereof can be done through high density nucleotide arrays as
described by Lockhart
et al.(1996) and Sosnowsky et a1.(1997). Oligonucleotides of 15-50 nucleotides
from the
sequences of the purH genomic DNA, the pzrrH cDNA sequences particularly those
comprising
at least one of biallelic markers according the present invention, preferably
at least one biallelic
marker selected from the group consisting of A I to A 17, A34 and A35, or the
sequences
complementary thereto, are synthesized directly on the chip (Lockhart et al.,
supra) or
synthesized and then addressed to the chip (Sosnowski et al., supra).
Preferably, the
oligonucleotides are about 20 nucleotides in length.
purH cDNA probes labeled with an appropriate compound, such as biotin,
digoxigenin
or fluorescent dye, are synthesized from the appropriate mRNA population and
then randomly
fragmented to an average size of 50 to 100 nucleotides. Tiie said probes are
then hybridized to
the chip. After washing as described in Lockhart et al., supra and application
of different
electric fields (Sosnowsky et al.. 1997)., the dyes or labeling compounds are
detected and
quantified. Duplicate hybridizations are performed. Comparative analysis of
the intensity of the
signal originating from cDNA probes on the same target oligonucleotide in
different cDNA
3
5 samples indicates a differential expression of purH mRNA.
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Methods For Inhibitin The Expression Of A purH Gene
Other therapeutic compositions according to the present invention comprise
advantageously an oligonucleotide fragment of the nucleic sequence of purH as
an antisense tool
or a triple helix tool that inhibits the expression of the corresponding pzrrH
gene. A preferred
fragment of the nucleic sequence of purH comprises an allele of at least one
of the biallelic
markers A l to A 17, A34 and A3 5.
Antisense Approach
Preferred methods using antisense polynucleotide according to the present
invention are
the procedures described by Sczakiel et al.(1995).
Preferably, the antisense tools are chosen among the polvnucleotides (15-200
bp long)
that are complementary to the 5'end of the purH mRNA. In another embodiment, a
combination
of different antisense polynucleotides complementary to different parts of the
desired targeted
gene are used.
Preferred antisense polynucleotides according to the present invention are
complementary to a sequence of the mRNAs of purH that contains either the
translation
initiation codon ATG or a splicing donor or acceptor site.
The antisense nucleic acids should have a length and melting temperature
sufficient to
permit formation of an intracellular duplex having sufficient stability to
inhibit the expression of
the ptrrH mRNA in the duplex. Strategies for designing antisense nucleic acids
suitable for use
in gene therapy are disclosed in Green et al., (1986) and Izant and Weintraub,
(1984), the
disclosures of which are incorporated herein by reference.
In some strategies, antisense molecules are obtained by reversing the
orientation of the
PURH coding region with respect to a promoter so as to transcribe the opposite
strand from that
which is normally transcribed in the cell. The antisense molecules may be
transcribed using in
vitro transcription systems such as those wliich employ T7 or SP6 polymerase
to generate the
transcript. Another approach involves transcription ofpurHantisense nucleic
acids in vivo by
operably linking DNA containing the antisense sequence to a promoter in a
suitable expression
vector.
Alternatively, suitable antisense strategies are those described by Rossi et
al.(1991), in
the International Applications Nos. WO 94/23026, WO 95/04141, WO 92/18522 and
in the
European Patent Application No. EP 0 572 287 A2, the disclosures of which are
incorporated by
reference herein in their entirety
An alternative to the antisense technology that is used according to the
present invention
consists in using ribozvmes that will bind to a target sequence via their
complementary
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polynucleotide tail and that will cleave the corresponding RNA by hydrolyzing
its target site
(namely "hammerhead ribozymes"). Briefly, the simplified cycle of a hammerhead
ribozyme
consists of (1) sequence specific binding to the target RNA via complementary
antisense
sequences; (2) site-specific hydrolysis of the cleavable motif of the target
strand; and (3) release
of cleavage products, which gives rise to another catalytic cycle. Indeed, the
use of long-chain
antisense polynucleotide (at least 30 bases long) or ribozymes with long
antisense arms are
advantageous. A preferred delivery system for antisense ribozyme is achieved
by covalently
linking these antisense ribozymes to lipophilic groups or to use liposomes as
a convenient
vector. Preferred antisense ribozymes according to the present invention are
prepared as
described by Sczakiel et al.(1995), the specific preparation procedures being
referred to in said
article being herein incorporated by reference.
Triple Helix Approach
The purH genomic DNA may also be used to inhibit the expression of the purH
gene
based on intracellular triple helix formation.
Triple helix oligonucleotides are used to inhibit transcription from a genome.
They are
particularly useful for studying alterations in cell activity when it is
associated with a particular
gene.
Similarly, a portion of the purH genomic DNA can be used to study the effect
of
inhibitingpurHtranscription within a cell. Traditionally, homopurine sequences
were
considered the most useful for triple helix strategies. However,
liomopyrimidine sequences can
also inhibit gene expression. Such homopyrimidine oligonucleotides bind to the
major groove at
liomopurine:homopyrimidine sequences. Thus, both types of sequences from the
purH genomic
DNA are contemplated within the scope of this invention.
To carry out gene therapy strategies using the triple helix approach, the
sequences of the
purH genomic DNA are first scanned to identify I 0-mer to 20-mer
homopyrimidine or
homopurine stretches which could be used in triple-helix based strategies for
inhibiting purH
expression. Following identification of candidate homopyrimidine or homopurine
stretches,
their efficiency in inhibiting purH expression is assessed by introducing
varying amounts of
oligonucleotides containing the candidate sequences into tissue culture cells
wliich express the
purH gene.
The oligonucleotides can be introduced into the cells using a variety of
inethods known
to those skilled in the art, inciuding but not limited to calcium phosphate
precipitation, DEAE-
Dextran, electroporation, liposome-inediated transfection or native uptake.
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Treated cells are monitored for altered cell function or reduced purH
expression using
techniques such as Northern blotting, RNase protection assays, or PCR based
strategies to
monitor the transcription levels of the parrH gene in cells which have been
treated with the
oligonucleotide.
The oligonucleotides which are effective in inhibiting gene expression in
tissue culture
cells may then be introduced in vivo using the techniques described above in
the antisense
approach at a dosage calculated based on the in vitro results, as described in
antisense approach.
In some embodiments, the natural (beta) anomers of the oligonucleotide units
can be
replaced with alpha anomers to render the oligonucleotide more resistant to
nucleases. Further,
an intercalating agent such as ethidium bromide, or the like, can be attached
to the 3' end of the
alpha oligonucleotide to stabilize the triple helix. For information on the
generation of
oligonucleotides suitable for triple helix formation see Griffin et al.(1989),
which is hereby
incorporated by this reference.
Computer-Related Embodiments
As used herein the term "nucleic acid codes of the invention" encompass the
nucleotide
sequences comprising, consisting essentiaily of, or consisting of any one of
the following: a) a
contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80,
90, 100, 150, 200, 500,
or 1000 nucleotides of SEQ ID No 1, wherein said contiguous span comprises at
least I of the
following nucleotide positions of SEQ ID No 1: 1-1587, 1729-2000, 2095-2414,
2558-3235,
3848-3991, 4156-7043, 7396-7958, 8237-9596, 9666-9874, 9921-10039, 1 0083-1 1
742, 11825-
15173, 1 5267-1 59 1 6, 16075-16750, 16916-22304. 22443-23269, 23384-24834,
24927-25952,
26048-28683, 28829-34694, 37282-37458, 37765-37894, 38563-38932, 39178-39451,
39692-
39821, 40038-40445, and 40846-41587; b) a contiguous span of at least 12, 15,
18, 20, 25, 30,
35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID
No 1, wherein said
contiguous span comprises a nucleotide selected from the group consisting of a
G at position
15234, and a G at position 36801of SEQ ID No 1; c) a contiguous span of at
least 12, 15, 18, 20,
25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of
SEQ ID No 2,
wherein said contiguous comprises a nucleotide selected in the group
consisting of a G at
position 424, and a G at position 1520 of SEQ ID No 2; d) a contiguous span of
at least 12, 15,
18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100. 150. 200, or 500 nucleotides,
to the extent that
such lenaths are consistent with the specific sequence, of a sequence selected
from the group
consisting of SEQ ID Nos. 4 to 22. and the complements thereof, optionally
wherein said
contiguous span comprises either allele I or allele 2 of apzrrH-related
biallelic marker selected
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from the group consisting of A18 to A33 and A36 to A43; and e) a nucleotide
sequence
complementary to any one of the preceding nucleotide sequences.
The "nucleic acid codes of the invention" further encompass nucleotide
sequences
homologous to a contiguous span of at least 30, 35, 40, 50, 60, 70, 80, 90,
100, 150, 200, 500, or
1000 nucleotides of the following nucleotide position range: 1-1587. 1729-
2000, 2095-2414,
2558-3235, 3848-3991, 4156-7043, 7396-7958, 8237-9596, 9666-9874. 9921-10039,
10083-
11742, 11825-15173, 15267-15916, 16075-16750, 16916-22304, 22443-23269, 23384-
24834,
24927-25952, 26048-28683, 28829-34694, 37282-37458, 37765-37894. 38563-38932,
39178-
39451, 39692-39821, 40038-40445, and 40846-41587 of SEQ ID No 1, and sequences
complementary to all of the preceding sequences. Homologous sequences refer to
a sequence
having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% homology to
these contiguous
spans. Homology may be determined using any method described herein. including
BLAST2N
with the default parameters or with any modified parameters. Homologous
sequences also may
include RNA sequences in which uridines replace the thymines in the nucleic
acid codes of the
invention. It will be appreciated that the nucleic acid codes of the invention
can be represented in
the traditional single character format (See the inside back cover of Stryer.
Lubert. Biochemistry, 3rd
edition. W. H Freeman & Co., New York.) or in any other format or code which
records the identity
of the nucleotides in a sequence.
As used herein the term "polypeptide codes of the invention" encompass the
polypeptide
sequences comprising a contiguous span of at least 6, 8, 10, 12, 15. 20, 25,
30, 40, 50, or 100
amino acids of SEQ ID No 3, wherein said contiguous span includes a serine
residue at amino
acid position 116 of SEQ ID No 3. It will be appreciated that the polypeptide
codes of the
invention can be represented in the traditional single character format or
three letter format (See the
inside back cover of Stryer, Lubert. Bioche-nistry, 3d edition. W. H Freeman &
Co., New York.) or
in any other format or code which records the identity of the polypeptides in
a sequence.
It will be appreciated by those skilled in the art that the nucleic acid codes
of the invention
and polypeptide codes of the invention can be stored. recorded, and
manipulated on any medium
which can be read and accessed by a computer. As used lierein, the words
"recorded" and "stored"
refer to a process for storing information on a computer medium. A skilled
artisan can readily adopt
any of the presently known methods for recording information on a computer
readable medium to
generate manufactures comprising one or more of the nucleic acid codes of the
invention, or one or
more of the polN-peptide codes of the invention. Another aspect of the present
invention is a
computer readable medium having recorded thereon at least 2. 5. 10, 15. 20.
25, 30. or 50 nucleic
acid codes of the invention. Another aspect of the present invention is a
computer readable
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medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, or 50
polypeptide codes of the
invention.
Computer readable media include magnetically readable media, optically
readable media,
electronically readable media and magnetic/optical media. For example, the
computer readable
media may be a hard disc, a floppy disc, a magnetic tape, CD-ROM, DVD, RAM, or
ROM as well
as other types of other media known to those skilled in the art.
Embodiments of the present invention include systems, particularly computer
systems
which contain the sequence information described herein. As used lierein, "a
computer system"
refers to the hardware components, software components, and data storage
components used to
store and/or analyze the nucleotide sequences of the nucleic acid codes of the
invention, the amino
acid sequences of the polypeptide codes of the invention, or other sequences.
The computer system
preferably includes the computer readable media described above, and a
processor for accessing and
manipulating the sequence data.
Preferably, the computer is a general purpose system that comprises a central
processing
unit (CPU), one or more data storage components for storing data, and one or
more data retrieving
devices for retrieving the data stored on the data storage components. A
skilled artisan can readily
appreciate that any one of the currently available computer systems are
suitable.
In one particular embodiment, the computer system includes a processor
connected to a bus
which is connected to a main memory, preferably implemented as RAM, and one or
more data
storage devices, such as a hard drive and/or other computer readable media
having data recorded
thereon. In some embodiments, the computer system further includes one or more
data retrieving
devices for reading the data stored on the data storage components. The data
retrieving device may
represent, for example, a floppy disk drive, a compact disk drive, a magnetic
tape drive, a hard disk
drive, a CD-ROM drive, a DVD drive, etc. In some embodiments, the data storage
component is a
removable computer readable medium such as a floppy disk, a compact disk, a
magnetic tape, etc.
containing control logic and/or data recorded thereon. The computer system may
advantageously
include or be programmed by appropriate software for reading the control logic
and/or the data from
the data storage component once inserted in the data retrieving device.
Software for accessing and
processing the nucleotide sequences of the nucleic acid codes of the
invention, or the amino acid
sequences of the polypeptide codes of the invention (such as search tools,
compare tools, modeling
tools, etc.) may reside in inain memory during execution.
In some embodiments, the computer system may further comprise a sequence
comparer for
comparing the nucleic acid codes of the invention or polypeptide codes of the
invention stored on a
computer readable medium to reference nucleotide or polypeptide sequences
stored on a computer
3 5 readable medium. A "sequence comparer" refers to one or more programs
Nvhich are implemented
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on the computer system to compare a nucleotide or polypeptide sequence with
other nucleotide or
polypeptide sequences and/or compounds including but not limited to peptides,
peptidomimetics,
and chemicals the sequences or structures of which are stored within the data
storage means. For
example, the sequence comparer mav compare the nucleotide sequences of the
nucleic acid codes of
the invention, or the amino acid sequences of the polypeptide codes of the
invention stored on a
computer readable medium to reference sequences stored on a computer readable
medium to
identify homologies, motifs implicated in biological function, or structural
motifs. The various
sequence comparer programs identified elsewhere in this patent specification
are particularly
contemplated for use in this aspect of the invention.
Accordingly, one aspect of the present invention is a computer system
comprising a
processor, a data storage device having stored thereon a nucleic acid code of
the invention or a
polypeptide code of the invention, a data storage device having retrievably
stored thereon
reference nucleotide sequences or polypeptide sequences to be compared to the
nucleic acid
code of the invention or polypeptide code of the invention and a sequence
comparer for
conducting the comparison. The sequence comparer may indicate a homology level
between the
sequences compared or identify structural motifs in the nucleic acid code of
the invention and
polypeptide codes of the invention or it may identify structural motifs in
sequences which are
compared to these nucleic acid codes and polypeptide codes. In some
embodiments, the data
storage device may have stored thereon the sequences of at least 2, 5, 10, 15,
20, 25, 30, or 50 of
the nucleic acid codes of the invention or polypeptide codes of the invention.
Another aspect of the present invention is a method for determining the level
of homology
between a nucleic acid code of the invention and a reference nucleotide
sequence, comprising the
steps of reading the nucleic acid code and the reference nucleotide sequence
through the use of a
computer program which determines homology levels and determining homology
between the
nucleic acid code and the reference nucleotide sequence with the computer
program. The computer
program may be any of a number of computer programs for determining homology
levels, including
those specifically enumerated herein, including BLAST2N with the default
parameters or with any
modified parameters. The method may be implemented using the computer systems
described
above. The method may also be performed by reading 2, 5, 10, 15, 20, 25, 30,
or 50 of the above
described nucleic acid codes of the invention through the use of the computer
program and
determining homology between the nucleic acid codes and reference nucleotide
sequences.
Alternatively, the computer program mav be a computer program which compares
the
nucleotide sequences of the nucleic acid codes of the present invention, to
reference nucleotide
sequences in order to determine wliether the nucleic acid code of the
invention differs from a
reference nucleic acid sequence at one or more positions. Optionally such a
program records the
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length and identity of inserted, deleted or substituted nucleotides with
respect to the sequence of
either the reference polynucleotide or the nucleic acid code of the invention.
In one embodiment,
the computer program may be a program which determines whether the nucleotide
sequences of the
nucleic acid codes of the invention contain one or more biallelic marker or
single nucleotide
polymorphisms (SNP) with respect to a reference nucleotide sequence. These
single nucleotide
polymorphisms may each comprise a single base substitution. insertion, or
deletion, while the
biallelic markers may each comprise nucleotide substitutions, insertions, or
deletions of 1 to 10
contiguous nucleotides, preferably I to 5 contiguous nucleotides.
Another aspect of the present invention is a method for determining the level
of homology
between a polypeptide code of the invention and a reference polypeptide
sequence, comprising the
steps of reading the polypeptide code of the invention and the reference
polypeptide sequence
through use of a computer program which determines homology levels and
determining homology
between the polypeptide code and the reference polypeptide sequence using the
computer program.
Accordingly, another aspect of the present invention is a method for
determining whether a
nucleic acid code of the invention differs at one or more nucleotides from a
reference nucleotide
sequence comprising the steps of reading the nucleic acid code and the
reference nucleotide
sequence through use of a computer program which identifies differences
between nucleic acid
sequences and identifying differences between the nucleic acid code and the
reference nucleotide
sequence with the computer program. In some embodiments, the computer program
is a program
which identifies single nucleotide polymorphisms. The method may be
implemented by the
computer systems described above. The method may also be performed by reading
at least 2, 5, 10,
15, 20, 25, 30" or 50 of the nucleic acid codes of the invention and the
reference nucleotide
sequences through the use of the computer program and identifying differences
between the nucleic
acid codes and the reference nucleotide sequences with the computer program.
In other embodiments the computer based system may further comprise an
identifier for
identifying features within the nueleotide sequences of the nucleic acid codes
of the invention or the
amino acid sequences of the polypeptide codes of the invention.
An "identifier" refers to one or more programs which identifies certain
features within
the above-described nucleotide sequences of the nucleic acid codes of the
invention or the amino
acid sequences of the polypeptide codes of the invention.
The nucleic acid codes of the invention or the polypeptide codes of the
invention may be
stored and manipulated in a variety of data processor programs in a variety of
formats. For
example, they may be stored as text in a word processing file, such as
MicrosoftWORD or
WORDPERFECT or as an ASCII file in a variety of database programs familiar to
those of skill in
the art, such as DB2. SYBASE, or ORACLE. In addition, many computer programs
and databases
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may be used as sequence comparers, identifiers, or sources of reference
nucleotide or polypeptide
sequences to be compared to the nucleic acid codes of the invention or the
polypeptide codes of
the invention. The following list is intended not to limit the invention but
to provide guidance to
programs and databases which are useful with the nucleic acid codes of the
invention or the
polypeptide codes of the invention. The programs and databases which may be
used include, but
are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications
Group),
GeneMine (Molecular Applications Group), Look (Molecular Applications Group),
MacLook
(Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX
(Altschul
et al, 1990), FASTA (Pearson and Lipman, 1988), FASTDB (Brutlag et al., 1990),
Catalyst
(Molecular Simulations Inc.), Catalyst/SHAPE (Molecular Simulations Inc.),
Cerius'.DBAccess
(Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.), Insight
II, (Molecular
Simulations Inc.). Discover (Molecular Simulations Inc.), CHARMm (Molecular
Simulations Inc.),
Felix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.),
QuanteMM, (Molecular
Simulations Inc.), Homology (Molecular Simulations Inc.), Modeler (Molecular
Simulations Inc.),
ISIS (Molecular Simulations Inc.), Quanta/Protein Design (Molecular
Simulations Inc.), WebLab
(Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations
Inc.), Gene
Explorer (Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.),
the
EMBL/Swissprotein database, the MDL Available Chemicals Directory database,
the MDL Drug
Data Report data base, the Comprehensive Medicinal Chemistry database,
Derwents's World Drug
Index database, the BioByteMasterFile database, the Genbank database, and the
Genseqn database.
Many other programs and data bases would be apparent to one of skill in the
art given the present
disclosure.
Throughout this application, various publications, patents, and published
patent
applications are cited. The disclosures of the publications, patents, and
published patent
specifications referenced in this application are hereby incorporated by
reference into the present
disclosure to more fully describe the state of the art to which this invention
pertains.
EXAMPLES
Example I
Identification Of Biallelic Markers - DNA Extraction
Donors were unrelated and healtliy. They presented a sufficient diversity for
being
representative of a French heterogeneous population. The DNA from 100
individuals was
extracted and tested for the detection of the biallefic markers.
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30 ml of peripheral venous blood were taken from each donor in the presence of
EDTA.
Cells (pellet) were collected after centrifugation for 10 minutes at 2000 rpm.
Red cells were
lysed by a lysis solution (50 ml final volume: 10 mM Tris pH7.6; 5 mM MgCl);
10 mM NaCI).
The solution was centrifuged (10 minutes, 2000 rpm) as many times as necessary
to eliminate
the residual red cells present in the supernatant, after resuspension of the
pellet in the lysis
solution.
The pellet of white cells was lysed overnight at 42 C with 3.7 mi of lysis
solution
composed of:
- 3 ml TE 10-2 (Tris-HCI 10 mM, EDTA 2 mM) / NaCI 0 4 M
- 200 l SDS 10%
- 500 l K-proteinase (2 mg K-proteinase in TE 10-2./ NaCl 0.4 M).
For the extraction of proteins, I mi saturated NaCl (6M) (1/3.5 v/v) was
added. After
vigorous agitation, the solution was centrifuged for 20 minutes at 10000 rpm.
For the precipitation of DNA, 2 to 3 volumes of 100% ethanol were added to the
previous supernatant, and the solution was centrifuged for 30 minutes at 2000
rpm. The DNA
solution was rinsed three times with 70% ethanol to eliminate salts, and
centrifuged for 20
minutes at 2000 rpm. The pellet was dried at 37 C, and resuspended in I ml TE
10-1 or 1 ml
water. The DNA concentration was evaluated by measuring the OD at 260 nm (1
unit OD = 50
gg/ml DNA).
To determine the presence of proteins in the DNA solution, the OD 260 / OD 280
ratio
was determined. Only DNA preparations having a OD 260 / OD 280 ratio between
1.8 and 2
were used in the subsequent examples described below.
The pool was constituted by mixing equivalent quantities of DNA from each
individual.
Example 2
Identification Of Biallelic Markers: Amplification Of Genomic DNA By PCR
The amplification of specific genomic sequences of the DNA samples of Example
I was
carried out on the pool of DNA obtained previously. In addition, 50 individual
samples were
similarly amplified.
PCR assays were performed using the following protocol:
Final volume 25 l
DNA 2 ng/ l
MgCI) 2 mM
dNTP (each) 200 gM
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primer (each) 2.9 ng/ 1
Ampli Taq Gold DNA polvmerase 0.05 unit/ l
PCR buffer (lOx = 0.1 M TrisHCl pH8.3 0.5M KCI) lx
Each pair of first primers was designed using the sequence information of the
purH gene
disclosed herein and the OSP software (Hillier & Green, 1991). This first pair
of primers was
about 20 nucleotides in length and had the sequences disclosed in Table I in
the columns labeled
"Position range of amplification primer in SEQ ID No 1," "Complementary
position range of
amplification primer in SEQ ID No I," "Position range of amplification
primer," and
"Complementary position range of amplification primer."
Table I
Amplicon Position Primer Position range of Primer Complementary
range of the name amplification primer name position range of
amplicon in in SEQ ID No 1 amplification primer
SEQ ID 1 in SEQ ID No 1
99-32284 6137 6597 BI 6137 6157 C1 6577 6597
99-5602 14864 15312 B2 14864 14882 C2 15292 15312
5-290 15837 16266 B3 15837 15855 C3 16249 16266
99-22573 16599 17049 B4 16599 16617 C4 17030 17049
99-22586 18131 18610 B5 18131 18150 C5 18592 18610
99-5596 22710 23149 B6 22710 22727 C6 23130 23149
5-293 23100 23530 B7 23100 23118 C7 23512 23530
5-294 25822 26241 B8 25822 25840 C8 26222 26241
99-23454 30332 30773 B9 30332 30352 C9 30754 30773
99-15528 30918 31408 B10 30918 30935 C10 31390 31408
99-15798 34780 35233 B11 34780 34799 Cll 35215 35233
5-297 36593 37036 B12 36593 36610 C12 37017 37036
99-32281 37060 37561 B13 37060 37080 C13 37541 37561
5-298 38946 39365 B14 38946 38965 C14 39346 39365
99-23460 39439 39886 B15 39439 39459 C15 39868 39886
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Amplicon Position Primer Position range of Primer Complementary
range of the name amplification primer name position range of
am licon amplification primer
99-22578 1 450 B16 1 18 C 16 430 450
SEQ ID No 4 SEQ ID No 4 SEQ ID No 4
99-22580 1 506 B17 1 18 C17 488 506
SEQIDNo5 SEQIDNo5 SEQIDNo5
99-22585 1 514 B18 1 21 C18 494 514
SEQ ID No 6 SEQ ID No 6 SEQ ID No 6
99-23437 1 497 B19 I 20 C19 478 497
SEQ ID No 7 SEQ ID No 7 SEQ ID No 7
99-23440 1 448 B20 1 21 C20 428 448
SEQ ID No 8 SEQ ID No 8 SEQ ID No 8
99-23442 1 457 B21 1 20 C21 437 457
SEQ ID No 9 SEQ ID No 9 SEQ ID No 9
99-23444 1 399 B22 1 19 C22 379 399
SEQ IDNo 10 SEQ IDNo 10 SEQ ID No 10
99-23451 I 547 B23 1 20 C23 529 547
SEQ ID No 11 SEQ ID No 1 1 SEQ ID No I 1
99-23452 1 400 B24 1 20 C24 380 400
SEQ ID No 12 SEQ ID No 12 SEQ ID No 12
99-28437 1 450 B25 1 20 C25 431 450
SEQ ID No 13 SEQ ID No 13 SEQ ID No 13
99-32278 1 494 B26 1 20 C26 474 494
SEQ ID No 14 SEQ ID No 14 SEQ ID No 14
99-5574 1 533 B27 1 20 C27 513 533
SEQ ID No 15 SEQ ID No 15 SEQ ID No 15
99-5575 1 472 B28 1 20 C28 452 472
SEQ ID No 16 SEQ ID No 16 SEQ ID No 16
99-5582 1 516 B29 1 19 C29 497 516
SEQ ID No 17 SEQ ID No 17 SEQ ID No 17
99-5590 1 461 B30 I 19 C30 441 461
SEQ ID No 18 SEQ ID No 18 SEQ ID No 18
99-5595 1 453 B31 1 18 C31 436 453
SEQ ID No 19 SEQ ID No 19 SEQ ID No 19
99-5604 1 467 B32 1 20 C32 447 467
SEQ ID No 20 SEQ ID No 20 SEQ ID No 20
99-5605 1 399 B33 1 18 C33 380 399
SEQ ID No 21 SEQ ID No 21 SEQ ID No 21
99-5608 1 529 B34 1 19 C34 509 529
SEQ ID No 22 SEQ ID No 22 SEQ ID No 22
Preferably, the primers contained a common oligonucleotide tail upstream of
the specific
bases taraeted for amplification which was useful for sequencing.
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Primers from the columns labeled "Position range of amplification primer in
SEQ ID No
1," and "Position range of amplification primer" contain the following
additional PU 5'
sequence: TGTAAAACGACGGCCAGT; and primers from the columns labeled
"Complementary position range of amplification primer in SEQ ID No 1," and
"Complementary
position range of amplification primer" contain the following RP 5' sequence:
CAGGAAACAGCTATGACC. The primer containing the additional PU 5' sequence is
listed in
SEQ ID No 23. The primer containing the additional RP 5' sequence is listed in
SEQ ID No 24.
The synthesis of these primers was performed following the phosphoramidite
method,
on a GENSET UFPS 24.1 synthesizer.
DNA amplification was performed on a Genius II thermocycler. After heating at
95 C
for 10 min, 40 cycles were performed. Each cycle comprised: 30 sec at 95 C, 54
C for I min,
and 30 sec at 72 C. For final elongation, 10 min at 72 C ended the
amplification. The
quantities of the amplification products obtained were determined on 96-well
microtiter plates,
using a fluorometer and Picogreen as intercalant agent (Molecular Probes).
Example 3
Identification Of Biallelic Markers - Seguencin2 Of Amplified Genomic DNA And
Identification Of Polymorphisms
The sequencing of the amplified DNA obtained in Example 2 was carried out on
ABI
377 sequencers. The sequences of the amplification products were determined
using automated
dideoxy terminator sequencing reactions with a dye terminator cycle sequencing
protocol. The
products of the sequencing reactions were run on sequencing gels and the
sequences were
determined using gel image analysis (ABI Prism DNA Sequencing Analvsis
software (2.1.2
version)).
The sequence data were further evaluated to detect the presence of biallelic
markers
within the amplified fragments. The polymorphism search was based on the
presence of
superimposed peaks in the electrophoresis pattern resulting from different
bases occurring at the
same position as described previously.
In the 30 fragments of amplification, 33 biallelic markers were detected. The
localization of these biallelic markers are as shown in Table 2.
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Table 2
Genic purH-related biallelic markers
Amplicon BM Marker Localization Polymor- BM position Position of Pro-
Name in purH gene hism in SEQ ID probes in bes
alll all2 No I No 2 SEQ ID No 1
99-32284 Al 99-32284-107 Intron 2 C T 6491 6479 6503 P I
99-5602 A2 99-5602-372 Exon 5 G C 15234 424 15222 15246 P2
TorS(116)
5-290 A3 5-290-32 Intron 5 C T 15868 15856 15880 P3
99-22573 A4 99-22573-321 Intron 6 C T 16729 16717 16741 P4
99-22586 A5 99-22586-300 Intron 7 G C 18311 18299 18323 P5
99-22586 A6 99-22586-39 Intron 7 C T 18572 18560 18584 P6
99-5596 A7 99-5596-216 (- Intron 8 A G 22906 22894 22918 P7
197)
5-293 A8 5-293-76 Intron 8 C T 23175 23163 23187 P8
5-293 A9 5-293-155 Intron 8 A G 23253 23241 23265 P9
5-294 A 10 5-294-285 Intron 11 G C 26106 26094 26118 P 10
99-23454 All 99-23454-317 Intron 12 A G 30464 30452 30476 P11
99-23454 A12 99-23454-105 Intron 12 G C 30669 30657 30681 P12
99-15528 A13 99-15528-333 Intron 12 A G 31250 31238 31262 P13
99-15798 A14 99-15798-86 Intron 13 A G 35148 35136 35160 P14
5-297 A15 5-297-209 Exon 14 A G 36801 1520 36789 36813 P15
99-32281 A34 99-32281-276 Intron 14 C T 37286 37274 37298 P33
99-32281 A35 99-32281-26 Intron 14 C T 37536 37524 37548 P34
5-298 A16 5-298-376 Intron 15 A G 39321 39309 39333 P16
99-23460 A17 99-23460-199 3'regul. region G T 39689 39677 39701 P17
Non- enic purH-related biallelic markers
Amplicon BM Marker Localization Polymor- BM position Position of Pro-
Name hism probes bes
alll a112
99-22578 A18 99-22578-78 Intergenic C T 78 in SEQ ID 66 90 P18
region No 4 SEQ ID No 4
99-22580 A19 99-22580-72 Intergenic A T 72 in SEQ ID 60 84 P19
region No 5 SEQ ID No 5
99-22585 A36 99-22585-462 Intergenic G C 462 in SEQ ID 450 474 P35
region No 6 SEQ ID No 6
99-23437 A20 99-23437-347 Intergenic A G 347 in SEQ ID 335 359 P20
region No 7 SEQ ID No 7
99-23440 A21 99-23440-274 Fibronectin A G 273 in SEQ ID 261 285 P21
gene No 8 SEQ ID No 8
99-23442 A22 99-23442-190 Fibronectin C T 190 in SEQ ID 178 202 P22
gene No 9 SEQ ID No 9
99-23442 A37 99-23442-396 Fibronectin A C 396 in SEQ ID 384 408 P36
gene No 9 SEQ ID No 9
99-23444 A23 99-23444-203 Fibronectin A G 203 in SEQ ID 191 215 P23
gene No 10 SEQ ID No 10
99-23451 A24 99-23451-78 Fibronectin A G 77 in SEQ ID 65 89 P24
gene No 11 SEQ ID No 11
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99-23451 A24 99-23451-78 Fibronectin A G 77 in SEQ ID 65 89 P24
gene No 11 SEQ ID No 11
99-23452 A25 99-23452-306 Fibronectin G T 306 in SEQ ID 294 318 P25
gene No 12 SEQ ID No 12
99-28437 A38 99-28437-417 Intergenic C T 417 in SEQ ID 405 429 P37
region No 13 SEQ ID No 13
99-32278 A39 99-32278-218 Intergenic A G 218 in SEQ ID 206 230 P38
region No 14 SEQ ID No 14
99-32278 A40 99-32278-414 Intergenic C T 414 in SEQ ID 402 426 P39
region No 14 SEQ ID No 14
99-5575 A26 99-5575-330 Intergenic C T 327 in SEQ ID 315 339 P26
region No 16 SEQ ID No 16
99-5582 A27 99-5582-71 Fibronectin G C 71 in SEQ ID 59 83 P27
gene No 17 SEQ ID No 17
99-5582 A41 99-5582-354 Fibronectin A G 354 in SEQ ID 342 366 P40
gene No 17 SEQ ID No 17
99-5590 A28 99-5590-99 Intergenic C T 99 in SEQ ID 87 111 P28
region No 18 SEQ ID No 18
99-5590 A42 99-5590-425 Intergenic G C 424 in SEQ ID 412 436 P41
region No 18 SEQ ID No 18
99-5595 A29 99-5595-380 Fibronectin A G 379 in SEQ ID 367 391 P29
gene No 19 SEQ ID No 19
99-5604 A30 99-5604-376 Fibronectin A G 374 in SEQ ID 362 386 P30
gene No 20 SEQ ID No 20
99-5605 A31 99-5605-90 Fibronectin G T 90 in SEQ ID 78 102 P31
gene No 21 SEQ ID No 21
99-5605 A43 99-5605-135 Fibronectin G T 135 in SEQ ID 123 147 P42
gene No 21 SEQ ID No 21
99-5608 A32 99-5608-324 Intergenic A G 323 in SEQ ID 311 335 P32
region No 22 SEQ ID No 22
99-5574 A33 99-5574-388 lntergenic Del 382 in SEQ ID
region AA No 15
BM refers to "biallelic marker". All1 and a112 refer respectively to allele I
and allele 2 of
the biallelic marker. "Freq. Of all2" refers to the frequency of the allele 2
in percentage in control
population. Frequencies corresponded to a population of random blood donors of
French Caucasian
origin.
Example 4
Validation Of The Polvmornhisms Throu$!h Microsequencinp_
The biallelic markers identified in Example 3 were further confirmed and their
respective
frequencies were determined througli microsequencing. Microsequencing was
carried out for each
individual DNA sample described in Example 1.
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Table 3
Marker Name Biallelic Mis. 1 Position range of Mis. 2 Complementary
Marker microsequencing position range of
primer mis. 1 in microsequencing
SEQ ID No 1 primer mis. 2 in
SEQ ID NO 1
99-32284-107 Al D I 6472 6490 El 6492 6510
99-5602-372 A2 D2 15215 15233 E2 15235 15253
5-290-32 A3 D3 15849 15867 E3 15869 15887
99-22573-321 A4 D4 16710 16728 E4 16730 16748
99-22586-300 A5 D5 18292 18310 E5 18312 18330
99-22586-39 A6 D6 18553 18571 E6 18573 18591
99-5596-216 A7 D7 22887 22905 E7 22907 22925
5-293-76 A8 D8 23156 23174 E8 23176 23194
5-293-155 A9 D9 23234 23252 E9 23254 23272
5-294-285 A10 D10 26087 26105 E10 26107 26125
99-23454-317 All DIi 30445 30463 E11 30465 30483
99-23454-105 A12 D12 30650 30668 E12 30670 30688
99-15528-333 A 13 D13 31231 31249 E13 31251 31269
99-15798-86 A14 D14 35129 35147 E14 35149 35167
5-297-209 A15 D15 36782 36800 E15 36802 36820
99-32281-276 A34 D16 37267 37285 E16 37287 37305
99-32281-26 A35 D17 37517 37535 E17 37537 37555
5-298-376 A16 D18 39302 39320 E18 39322 39340
99-23460-199 A17 D19 39670 39688 E19 39690 39708
Marker Name BM Mis. 1 Position range of Mis. 2 Complementary
microsequencing position range of
primer microsequencing
primer
99-22578-78 A18 D20 59 77 E20 79 97
SEQ ID No 4 SEQ ID No 4
99-22580-72 A19 D21 53 71 E21 73 91
SEQ ID No 5 SEQ ID No 5
99-22585-462 A36 D22 443 461 E22 463 481
SEQ ID No 6 SEQ ID No 6
99-23437-347 A20 D23 328 346 E23 348 366
SEQIDNo7 SEQIDNo7
99-23440-274 A21 D24 254 272 E24 274 292
SEQ ID No 8 SEQ ID No 8
99-23442-190 A22 D25 171 189 E25 191 209
SEQ ID No 9 SEQ ID No 9
99-23442-396 A37 D26 377 395 E26 397 415
SEQIDNo9 SEQIDNo9
99-23444-203 A23 D27 184 202 E27 204 222
SEQ ID No 10 SEQ ID No 10
99-23451-78 A24 D28 58 76 E28 78 96
SEQIDNo 11 SEQIDNo Ii
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99-23452-306 A25 D29 287 305 E29 307 325
SEQ ID No 12 SEQ ID No 12
99-28437-417 A38 D30 398 416 E30 418 436
SEQ ID No 13 SEQ ID No 13
99-32278-218 A39 D31 199 217 E31 219 237
SEQ ID No 14 SEQ ID No 14
99-32278-414 A40 D32 395 413 E32 415 433
SEQ ID No 14 SEQ ID No 14
99-5575-330 A26 D33 308 326 E33 328 346
SEQ ID No 16 SEQ ID No 16
99-5582-71 A27 D34 52 70 E34 72 90
SEQ ID No 17 SEQ ID No 17
99-5582-354 A41 D35 335 353 E35 355 373
SEQ ID No 17 SEQ ID No 17
99-5590-99 A28 D36 80 98 E36 100 118
SEQ ID No 18 SEQ ID No 18
99-5590-425 A42 D37 405 423 E37 425 443
SEQ ID No 18 SEQ ID No 18
99-5595-380 A29 D38 360 378 E38 380 398
SEQ ID No 19 SEQ ID No 19
99-5604-376 A30 D39 355 373 E39 375 393
SEQ ID No 20 SEQ ID No 20
99-5605-90 A31 D40 71 89 E40 91 109
SEQ ID No 21 SEQ ID No 21
99-5605-135 A43 D41 116 134 E41 136 154
SEQ ID No 21 SEQ ID No 21
99-5608-324 A32 D42 303 322 E42 324 343
SEQ ID No 14 SEQ ID No 14
Amplification from genomic DNA of individuals was performed by PCR as
described
above for the detection of the biallelic markers with the same set of PCR
primers (Table 1).
The preferred primers used in microsequencing were about 19 nucleotides in
length and
hybridized just upstream of the considered polymorphic base. According to the
invention, the
primers used in microsequencing are detailed in Table 3.
The microsequencing reaction was performed as follows
After purification of the amplification products, the microsequencing reaction
mixture
was prepared by adding, in a 20 l final volume: 10 pmol microsequencing
oligonucleotide, l U
Thermosequenase (Aniersham E79000G), 1.25 l Thermosequenase buffer (260 mM
Tris HCI
pH 9.5. 65 mM MgCl2), and the two appropriate fluorescent ddNTPs (Perkin
Elmer, Dye
Terminator Set 401095) complementary to the nucleotides at the polymorphic
site of each
biallelic marker tested, following the manufacturer's recommendations. After 4
minutes at
94 C, 20 PCR cycles of 15 sec at 55 C, 5 sec at 72 C, and 10 sec at 94 C were
carried out in a
Tetrad PTC-225 tliermocycler (MJ Research). The unincorporated dye terminators
were then
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removed by ethanol precipitation. Samples were finally resuspended in
formamide-EDTA
loading buffer and heated for 2 min at 95 C before being loaded on a
polyacrylamide sequencing
gel. The data were collected by an ABI PRISM 377 DNA sequencer and processed
using the
GENESCAN software (Perkin Elmer).
Following gel analysis, data were automatically processed with software that
allows the
determination of the alleles of biallelic markers present in each amplified
fragment.
The software evaluates such factors as whether the intensities of the signals
resulting
from the above microsequencing procedures are weak, normal, or saturated, or
whether the
signals are ambiguous. In addition, the software identifies significant peaks
(according to shape
and height criteria). Among the significant peaks, peaks corresponding to the
targeted site are
identified based on their position. When two significant peaks are detected
for the same
position, each sample is categorized classification as homozygous or
heterozygous type based on
the height ratio.
Example 5
Association study between prostate cancer and the purH-related biallelic
markers
Collection of DNA samples from affected and non-affected individuals
Affected population:
The positive trait followed in this association study was prostate cancer.
Prostate cancer
patients were recruited according to a combination of clinical, histological
and biological
inclusion criteria. Clinical criteria can include rectal examination and
prostate biopsies.
Biological criteria can include PSA assays. The affected individuals were
recorded as familial
forms when at least two persons affected by prostate cancer have been
diagnosed in the family.
Familial forms in which at least three persons are affected by prostate cancer
in the family are
described in the present application as >3CaP. Remaining cases were classified
as informative
sporadic cases when at least two sibs of the case both aged over 50 years old
are unaffected, or
uninformative sporadic cases when no information about sibs over 50 years old
is available. All
affected individuals included in the statistical analysis of this patent were
unrelated. Cases were
also separated followitig the criteria of diagnosis age: early onset prostate
cancer (under 65 years
old) and late onset prostate cancer (65 years old or more).
Unaffected population:
Control individuals included in this study were checked for both the absence
of all
clinical and biological criteria defining the presence or the risk of prostate
cancer (PSA < 4)
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(WO 96/21042), and for their age (aged 65 years old or more). All unaffected
individuals
included in the statistical analysis of this patent were unrelated.
The affected group was composed of 491 unrelated individuals, comprising 197
familial
cases and 294 sporadic cases. Among the familial cases, 85 individuals are
>3CaP. Among the
sporadic cases, 70 individuals are informative sporadic cases. The unaffected
group contained
313 individuals aged 65 years old or more.
Genotyping of affected and control individuals
The general strategy to perform the Association studies was to individually
scan the
DNA samples from all individuals in each of the populations described above in
order to
establish the allele frequencies of the above described biallelic markers in
each of these
populations.
Allelic frequencies of the above-described biallelic marker alleles in each
population
were determined by performing microsequencing reactions on amplified fragments
obtained by
genomic PCR performed on the DNA samples from each individual. Genomic PCR and
microsequencing were performed as detailed above in Examples I and 2 using the
described
PCR and microsequencing primers.
Haplotype frequency analysis
None of the single marker alleles showed a significant association with
prostate cancer
except the biallelic marker 99-23437/347 (A20) in the informative sporadic
individuals (p value
of 1.9 10"3). However, significant results were obtained in haplotype studies.
Allelic
frequencies were useful to check that the markers used in the haplotype
studies meet the Hardy-
Weinberg proportions (random mating).
For sets of 2 and 3 markers haplotype frequency estimation can be derived
using the E-
M algorithm (see above). It has to be noted that all of these approaches are
applied to markers
under Hardy-Weinberg equilibrium, and only these markers are included.
The profile of haplotypes frequencies can be compared by two main approaches.
Omnibus Likelihood ratio tests
For one combination of 2 and 3 markers, this procedure allows us to compare
the profile
of haplotype frequency differences between the two populations under study.
The null
hypothesis is tilat both cases and controls are samples derived from the same
population, i.e., the
haplotypes frequencies are only due to the sampling process. Using the E-M
algorithm, one can
calculate the haplotype frequencies in cases. in controls and in the overall
population. Once the
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haplotype frequencies are estimated, a likelihood ratio test (LR test) can be
derived which
gathers all the haplotype frequency differences in one statistic.
As 1) haplotype frequencies are inferred via the E-M algorithm and not
observed and 2)
that rare haplotypes occur, the LR test does not follow a chi-square with h-I
degrees of freedom
(h being the number of haplotypes). A permutation procedure then allows
assessment of the
significance of the LR test. The permutation procedure is performed as follows
:
The affected status (case/controls) is shuffled in individuals and replicate
samples of
original size are generated. For each generated replicate sample haplotype
frequencies are
derived and a LR test is calculated. This procedure mimics the null hypothesis
of the test, i.e. the
two samples are derived from a single population. The process is repeated
generally a hundred
times. The proportion of test superior to the observed value (the real value)
is the level of
significance of the test.
Haplo-max test
Another procedure is based on the haplotype frequency difference of each
haplotype
between the two groups. For one combination of marker with h haplotypes, h
differences of
haplotype frequencies can be compared via a Pearson chi-square statistic (1
degree of freedom).
The haplo-max test selects the difference showing the maximum positive (Max-M)
or negative
(Max-S) test value between cases and controls, rejecting test values based on
rare haplotype
frequencies (with an estimated number of haplotypes inferior to 10). Here, for
one combination
of marker, there is one Max-M and one Max-S test value.
The significance of this test can be compared by several means
- First, significance thresholds taking into account the multiple testing
procedure due to
selection of the maximum test value can be arbitrarily set,
- Secondly, one can assess the observed distribution of the statistics based
on all Max-M
(or Max-S) statistics derived from the analysis and estimate signification
thresholds,
- Thirdly, one can use the permutation procedure to evaluate a level of
significance not
based on chi-square with one degree of freedom.
The results of the haplotype analysis using 20 biallelic markers (99-23437/347
(A20),
99-5605/90 (A31), 99-23452/306 (A25), 99-5604/376 (A30), 99-23440/274 (A21),
99-5582/71
(A27), 99-23451/78 (A24). 99-23442/190 (A22), 99-23444/203 (A23), 99-5595/380
(A29), 99-
5608/324 (A32). 99-23460/199 (A17) , 99-15798/86 (A14), 99-15528/333 (A13), 99-
23454/317
(A11), 99-5596/216 (A7), 99-22573/321 (A4), 99-5602/372 (A2), 99-5575/330
(A26), and 99-
5590/99 (A28)) are shown in Figures 1 and 2. Haplotype analysis for
association of purH-
related biallelic markers and prostate was performed by estimating the
frequencies of all possible
2, 3 and 4-narker haplotvpes in the affected and control populations described
above.
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Haplotype estimations were performed by applying the Expectation-Maximization
(EM)
algorithm (Excoffier and Slatkin, 1995), using the EM-HAPLO program (Hawley et
al., 1994) as
described above. Estimated haplotype frequencies in the affected and control
population were
compared by means of a chi-square statistical test (one degree of freedom).
Sporadic cases
Figure 1 shows the most significant haplotypes obtained with the sporadic
cases.
Haplotype no.l (HAP 1) consisting of two biallelic markers (99-5595/380 (A29)
allele A,
and 99-5596/216 (A7) allele A, presented a p-value of 1.1x10-9 and an odd-
ratio of 22.
Estimated haplotype frequencies were 6.9% in the sporadic cases and 0.3 % in
the controls. The
association between the HAP 1 haplotype and prostate cancer was still more
significant in the
sporadic cases under 65 years old with a p-value of 2x10-13 (see figure 3)
However, six other two-markers haplotypes are also hiahly significant, namely
HAP2,
HAP3, HAP4, HAP5, HAP6, and HAP7. These haplotypes presented p-value comprised
in the
range between 2.2x10-8 and 8.3x10-'. They often comprised the biallelic marker
(99-5596/216
(A7) allele A. Haplotype HAP8 had a highly significant p value in the
informative sporadic
population (2.6x10-7) (see Figure 3).
Haplotype no.9 (14AP9) consisting of three biallelic markers (99-23444/203
(A23) allele
G, 99-5595/380 (A29) allele A and 99-5596/216 (A7) allele A, had a p-value of
3x10-8 and an
odd ratio of 18.64. Estimated haplotype frequencies were 6.5 % in the cases
and 0.4% in the
controls. The three-markers haplotypes HAP 10 to HAP 17 and the four-markers
haplotypes
HAP20 to HAP28 also showed very significant association. The haplotypes HAP10
to HAP17
and HAP20 to HAP28 all comprise the biallelic marker 99-5596/216 (A7).
The more preferred haplotypes HAP 1 and HAP9 are both strongly associated with
sporadic prostate cancer. They can be used in diagnosis of prostate cancer.
The statistical significance of the results obtained for the haplotype
analysis was
evaluated by a phenotypic permutation test reiterated 1000 times on a
computer. For this
computer simulation. data from the affected and control individuals were
pooled and randomly
allocated to two groups which contained the same number of individuals as the
case-control
populations used to produce the data summarized in figure 1. A haplotype
analysis was then run
on these artificial groups for the 2 markers included in the haplotype HAP 1
and in the haplotype
HAP8 which. showed the strongest association with sporadic prostate cancer,
more particularly
with informative sporadic prostate cancer for the HAP8. This experiment was
reiterated 1000
times and the results are shown in figure 3. These results demonstrate for the
HAPI haplotype
of figure 1 that amon- 1000 iterations none of the obtained haplotypes in the
simulation had a p-
value comparable to the one obtained for the haplotype FiAPI. These results
clearlv validate the
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statistical significance of the association between the HAPl haplotype and
prostate cancer,
preferably sporadic prostate cancer. The permutation test also shows for the
HAP8 of figure 1
haplotype that among 1000 iterations none of the obtained haplotypes in the
simulation had a p-
value comparable to the one obtained for the haplotype HAP8 with the
informative sporadic
cases.
Haplotvpe analysis with genic biallelic markers of the purH gene
The results of the haplotype analysis using 7 biallelic markers (5-297-209
(A15), 99-
15798-86 (A14), 99-15528-333 (A13), 5-294-285 (A10), 99-5596-216 (A7), 99-
22573-321
(A4), and 99-5602-372 (A2)) are shown in Figures 4 and 5. Haplotype analysis
for association
of genic purH-related biallelic markers and prostate was performed by
estimating the
frequencies of all possible 2, and 3 marker haplotypes in the affected.and
control populations
described above.
Figure 4 shows the most significant haplotypes obtained with the sporadic
cases (Figure
4A : 2-markers haplotypes ; Figure 4B : 3-markers haplotypes).
Two 2-biallelic markers haplotypes, namely Haplotype no I and 2, showed a
highly
significant association with sporadic prostate cancer.
Haplotype no.l (HAP1) consisting of two biallelic markers (5-294-285 (A10)
allele G,
and 99-5596-216 (A7) allele A), presented for the haplotype frequency test a p-
value of 2.8x10-'
and an odd-ratio of 100. Estimated haplotype frequencies were 4.5 % in the
sporadic cases and
0 % in the controls. This haplotype presented a p-value for the likelihood
ratio test of 3.2 x 10-
7 .The association between the HAP I haplotype and prostate cancer was still
more significant in
the sporadic cases under 65 years old with a p-value of 1.9x10-8 and in the
informative sporadic
cases with a p-value of 1.2x10-11 (see figure 5).
Haplotype no.2 (HAP2) consisting of two biallelic markers (99-15528-333 (A13)
allele
G, and 99-5596-216 (A7) allele A), presented for the haplotype frequency test
a p-value of lxl0"
6 and an odd-ratio of 100. Estimated haplotype frequencies were 3.9 % in the
sporadic cases and
0 % in the controls. This haplotype presented a p-value for the likelihood
ratio test of 1.1 x 10-5.
Two 3-biallelic markers haplotypes, namely Haplotype no 18 and 19, showed a
highly
significant association with sporadic prostate cancer. Compared to the 2-
markers haplotypes,
these 3-markers haplotypes further comprise the biallelic marker 5-297-209 (A
15), allele A.
Haplotype no.18 (HAP18) consisting ofthree biallelic markers (5-294-285 (A10)
allele
G, 99-5596-216 (A7) allele A and 5-297-209 (A 15), allele A), presented for
the haplotype
frequency test a p-value of 3.8x10-' and an odd-ratio of 100. Estimated
haplotype frequencies
were 4.5 % in the sporadic cases and 0 % in the controls. This haplotype
presented a p-value for
the likelihood ratio test of 3.5 x 10-6.
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Haplotype no.19 (HAP19) consisting of three biallelic markers (99-15528-333
(A13)
allele G, 99-5596-216 (A7) allele A and 5-297-209 (A15), allele A), presented
for the haplotype
frequency test a p-value of 1.2x10-6 and an odd-ratio of 100. Estimated
haplotype frequencies
were 4 % in the sporadic cases and 0 % in the controls. This haplotype
presented a p-value for
the likelihood ratio test of 1.1 x 10"4
.
The more preferred haplotypes HAP 1 and HAP2 are both strongly associated with
sporadic prostate cancer. They can be used in diagnosis of prostate cancer.
The statistical significance of the results obtained for the haplotype
analysis was
evaluated by a phenotypic permutation test reiterated 1000 times on a
computer. The
permutation tests demonstrate for the HAP 1, HAP2, HAP 18 and HAP 19
haplotypes of figure 4
that among 100 iterations none of the obtained haplotypes in the simulation
had a p-value
comparable to the one obtained for these haplotypes. Moreover, the permuation
test for the
HAP1 haplotype of Figure 4 demonstrates that among 1000 iterations none of the
obtained
haplotypes in the simulation had a p-value comparable to the one obtained for
the HAPI
haplotype for the sporadic cases. These results clearly validate the
statistical significance of the
association between the HAP 1, HAP2, HAP 18 and HAP 19 haplotypes of Figure 4,
more
particularly HAP1 haplotype, and prostate cancer, preferably sporadic prostate
cancer.
HAP1, HAP2, HAP18 and HAP19 haplotypes of Figure 4, preferably HAP1 haplotype,
can be used in diagnosis of prostate cancer, more particularly sporadic
prostate cancer.
Familial cases
Figure 2 shows the most significant haplotypes obtained with the familial
cases.
Two three-markers haplotypes, namely HAP9 and HAP 10, showed a highly
significant
association with familial prostate cancer. The haplotype HAP9 consisting of
three biallelic
markers (99-5605/90 allele G, 99-23460/199 (Al7) allele C and 99-5590/99 (A28)
allele T,
presented a p-value of 2.1x10-' and an odd-ratio of 2.43. Estimated haplotype
frequencies were
16.8% in the familial cases and 7.6 % in the controls. The haplotype HAP 10
consisting of three
biallelic markers (99-5604/376 (A30) allele G. 99-23460/199 (Al7) allele C and
99-5590/99
(A28) allele T, presented a p-value of 3.7x 10- and an odd-ratio of 2.32.
Estimated haplotype
frequencies were 17.1% in the familial cases and 8.2 % in the controls. The
association between
the HAP 10 haplotype and prostate cancer was more significant in the familial
cases which are
either >=3CaP or under 65 years old with a p-value of 1.4x10-' or 7.1 x 10-' ,
respectively (see
figure 3). However, ten other three-markers haplotypes are also significant.
namely HAP11 to
HAP20. These haplotypes presented p-value comprised in the range between
8.3x10-' and
9.6x 10-'.
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131
The four-markers haplotypes HAP 22 to HAP 33 showed a highly significant
association
wih familial prostate cancer and presented p-values comprised in the range
between 3.2x10-' and
9.5x10-6. One preferred haplotype HAP22 consisting of the four biallelic
markers (99-
23452/306 (A25) allele G, 99-5582/71 (A27) allele G, 99-15798/86 (A14) allele
T and 99-
5590/99 (A28) allele T, presented a p-value of 3.2x10-' and an odd-ratio of
2.82. Estimated
haplotype frequencies were 18.6 % in the familial cases and 7.5 % in the
controls. An other
preferred haplotype HAP 24 consisting of the four biallelic markers (99-
23452/306 (A25) allele
G, 99-23440/274 (A21) allele A, 99-15798/86 (A 14) allele T and 99-5590/99
(A28) allele T,
presented a p-value of 1x10-6 and an odd-ratio of 2.73. Estimated haplotype
frequencies were
18.6 % in the familial cases and 7.7 % in the controls. The association
between the HAP 24
haplotype and prostate cancer was still more significant in the familial cases
which are either
>=3CaP or under 65 years old with a p-value of 9.Ix10-1 1 or 3.5x10-9 ,
respectively (see figure
3).
The haplotypes HAP10 and HAP24 are the more preferred haplotype of the
invention. It
can be used in diagnosis of prostate cancer and more particularly familial
prostate cancer.
The statistical significance of the results obtained for the haplotype
analysis was
evaluated by a phenotypic permutation test reiterated 1000 times on a
computer. For this
computer simulation, data from the affected and control individuals were
pooled and randomly
allocated to two groups which contained the same number of individuals as the
case-control
populations used to produce the data summarized in figure 2. A haplotype
analysis was then run
on these artificial groups for the 3 markers included in the haplotype HAP10
and for the 4
markers included in the haplotype HAP24 which, showed the strongest
association with familial
prostate cancer. more particularly with prostate cancer >=3CaP or under 65
years old. This
experiment was reiterated 1000 times and the results are shown in figure 3.
These results
demonstrate for the HAP 10 haplotype that among 1000 iterations none or only
one of the
obtained haplotypes had a p-value comparable to the one obtained for the
haplotype HAP10 with
the familial cases, and more particularly familial cases >=3CaP or under 65
years old. The
permutation test also shows for the HAP24 haplotype that among 1000 iterations
none of the
obtained haplotypes had a p-value comparable to the one obtained for the
haplotype HAP24 with
the familial cases, and more particularly familial cases >=3CaP or under 65
years old. These
results clearlv validate the statistical sianificance of the association
between the HAP 10 and
HAP24 haplotypes and prostate cancer. more particularly familial prostate
cancer and more
preferably either >=3CaP familial prostate cancer or familial prostate cancer
under 65 years old.
All references cited herein are incorporated by reference herein in their
entirety
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REFERENCES
The following references are cited herein and are incorporated herein by
reference in
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SEQUENCE LISTING FREE TEXT
The following free text appears in the accompanying Sequence Listing :
5'regulatory region
3'regulatory region
polymorphic base
or
complement
probe
deletion
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insertion
sequencing oligonucleotide Primer
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<110> Genset
<120> Genomic sequence of the purH gene and purH-related biallelic markers.
<130> 61.W01
<150> US 60/125,961
<151> 1999-03-24
<160> 24
'<170> Patent.pm
<210> 1
<211> 41684
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<222> 1..2000
<223> 5'regulatory region
<220>
<221> exon
<222> 2001..2096
<223> exon 1
<220>
<221> exon
<222> 2433..2559
<223> exon 2
<220>
<221> exon
<222> 8092..8168
<223> exon 3
<220>
<221> exon
<222> 9600..9666
<223> exon 4
<220>
<221> exon
<222> 15178..15266
<223> exon 5
<220>
<221> exon
<222> 15924..16075
<223> exon 6
<220>
<221> exon
<222> 16759..16915
<223> exon 7
<220>
<221> exon
<222> 22309..22434
<223> exon 8
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<220>
<221> exon
<222> 23277..23384
<223> exon 9
<220>
<221> exon
<222> 24841..24926
<223> exon 10
<220>
<221> exon
<222> 25957..26046
<223> exon 11
<220>
<221> exon
<222> 28700..28828
<223> exon 12
<220>
<221> exon
<222> 34699..34791
<223> exon 13
<220>
<221> exon
<222> 36679..36861
<223> exon 14
<220>
<221> exon
<222> 39014..39169
<223> exon 15
<220>
<221> exon
<222> 39456..39684
<223> exon 16
<220>
<221> miscfeature
<222> 39685..41684
<223> 3'regulatory region
<220>
<221> allele
<222> 6491
<223> 99-32284-107 : polymorphic base C or T
<220>
<221> allele
<222> 15234
<223> 99-5602-372 : polymorphic base G or C
<220>
<221> allele
<222> 15868
<223> 5-290-32 : polymorphic base C or T
<220>
<221> allele
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<222> 16729
<223> 99-22573-321 : polymorphic base C or T
<220>
<221> allele
<222> 18311
<223> 99-22586-300 polymorphic base G or C
<220>
<221> allele
<222> 18572
<223> 99-22586-39 : polymorphic base C or T
<220>
<221> allele
<222> 22906
<223> 99-5596-197 : polymorphic base A or G
<220>
<221> allele
<222> 23175
<223> 5-293-76 : polymorphic base C or T
<220>
<221> allele
<222> 23253
<223> 5-293-155 : polymorphic base A or G
<220>
<221> allele
<222> 26106
<223> 5-294-285 : polymorphic base G or C
<220>
<221> allele
<222> 30464
<223> 99-23454-317 polymorphic base A or G
<220>
<221> allele
<222> 30669
<223> 99-23454-105 : polymorphic base G or C
<220>
<221> allele
<222> 31250
<223> 99-15528-333 polymorphic base A or G
<220>
<221> allele
<222> 35148
<223> 99-15798-86 : polymorphic base A or G
<220>
<221> allele
<222> 36801
<223> 5-297-209 : polymorphic base A or G
<220>
<221> allele
<222> 37286
<223> 99-32281-276 : polymorphic base C or T
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<220>
<221> allele
<222> 37536
<223> 99-32281-26 : polymorphic base C or T
<220>
<221> allele
<222> 39321
<223> 5-298-376 : polymorphic base A or G
<220>
<221> allele
<222> 39689
<223> 99-23460-199 : polymorphic base G or T
<220>
<221> primer_bind
<222> 6137..6157
<223> 99-32284.rp
<220>
<221> primer_bind
<222> 6577..6597
<223> 99-32284.pu complement
<220>
<221> primer_bind
<222> 14864..14882
<223> 99-5602.pu
<220>
<221> primerbind
<222> 15292._15312
<223> 99-5602.rp complement
<220>
<221> primer_bind
<222> 15837..15855
<223> 5-290.pu
<220>
<221> primerbind
<222> 16249._16266
<223> 5-290.rp complement
<220>
<221> primer_bind
<222> 16599..16617
<223> 99-22573.rp
<220>
<221> primerbind
<222> 17030._17049
<223> 99-22573.pu complement
<220>
<221> primerbind
<222> 18131._18150
<223> 99-22586.rp
<220>
<221> primer_bind
<222> 18592..18610
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<223> 99-22586."pu complement
<220>
<221> primerbind
<222> 22710..22727
<223> 99-5596.pu
<220>
<221> primerbind
<222> 23100..23118
<223> 5-293.pu
<220>
<221> primer_bind
<222> 23130..23149
<223> 99-5596.rp complement
<220>
<221> primer_bind
<222> 23512..23530
<223> 5-293.rp complement
<220>
<221> primerbind
<222> 25822._25840
<223> 5-294.pu
<220>
<221> primer_bind
<222> 26222..26241
<223> 5-294.rp complement
<220>
<221> primer_bind
<222> 30332..30352
<223> 99-23454.rp
<220>
<221> primer_bind
<222> 30754..30773
<223> 99-23454.pu complement
<220>
<221> primerbind
<222> 30918._30935
<223> 99-15528.pu
<220>
<221> primer_bind
<222> 31390..31408
<223> 99-15528.rp complement
<220>
<221> primer_bind
<222> 34780..34799
<223> 99-15798.rp
<220>
<221> primerbind
<222> 35215._35233
<223> 99-15798.pu complement
<220>
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
6
<221> primerbind
<222> 36593._36610
<223> 5-297.pu
<220>
<221> primer_bind
<222> 37017..37036
<223> 5-297.rp complement
<220>
<221> primer_bind
<222> 37060..37080
<223> 99-32281.rp
<220>
<221> primer_bind
<222> 37541..37561
<223> 99-32281.pu complement
<220>
<221> primer_bind
<222> 38946..38965
<223> 5-298.pu
<220>
<221> primer_bind
<222> 39346..39365
<223> 5-298.rp complement
<220>
<221> primerbind
<222> 39439._39459
<223> 99-23460.rp
<220>
<221> primerbind
<222> 39868._39886
<223> 99-23460.pu complement
<220>
<221> primer_bind
<222> 6472..6490
<223> 99-32284-107.mis
<220>
<221> primer_bind
<222> 6492..6510
<223> 99-32284-107.mis complement
<220>
<221> primerbind
<222> 15215._15233
<223> 99-5602-372.mis
<220>
<221> primerbind
<222> 15235._15253
<223> 99-5602-372.mis complement
<220>
<221> primer_bind
<222> 15849..15867
<223> 5-290-32.mis
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
7
<220>
<221> primerbind
<222> 15869..15887
<223> 5-290-32.mis complement
<220>
<221> primerbind
<222> 16710._16728
<223> 99-22573-321.mis
<220>
<221> primer_bind
<222> 16730..16748
<223> 99-22573-321.mis complement
<220>
<221> primer_bind
<222> 18292..18310
<223> 99-22586-300.mis
<220>
<221> primer_bind
<222> 18312..18330
<223> 99-22586-300.mis complement
<220>
<221> primer_bind
<222> 18553..18571
<223> 99-22586-39.mis
<220>
<221> primer_bind
<222> 18573..18591
<223> 99-22586-39.mis complement
<220>
<221> primer_bind
<222> 22887..22905
<223> 99-5596-197.mis
<220>
<221> primer_bind
<222> 22907..22925
<223> 99-5596-197.mis complement
<220>
<221> primer_bind
<222> 23156..23174
<223> 5-293-76.mis
<220>
<221> primerbind
<222> 23176._23194
<223> 5-293-76.mis complement
<220>
<221> primer bind
<222> 23234._23252
<223> 5-293-155.mis
<220>
<221> primer bind
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
8
<222> 23254..23272
<223> 5-293-155.mis complement
<220>
<221> primerbind
<222> 26087..26105
<223> 5-294-285.mis
<220>
<221> primer_bind
<222> 26107..26125
<223> 5-294-285.mis complement
<220>
<221> primerbind
<222> 30445._30463
<223> 99-23454-317.mis
<220>
<221> primer_bind
<222> 30465..30483
<223> 99-23454-317.mis complement
<220>
<221> primer_bind
<222> 30650..30668
<223> 99-23454-105.mis
<220>
<221> primer_bind
<222> 30670..30688
<223> 99-23454-105.mis complement
<220>
<221> primer_bind
<222> 31231..31249
<223> 99-15528-333.mis
<220>
<221> primer_bind
<222> 31251..31269
<223> 99-15528-333.mis complement
<220>
<221> primerbind
<222> 35129._35147
<223> 99-15798-86.mis
<220>
<221> primerbind
<222> 35149._35167
<223> 99-15798-86.mis complement
<220>
<221> primer_bind
<222> 36782..36800
<223> 5-297-209.mis
<220>
<221> primer_bind
<222> 36802..36820
<223> 5-297-209.mis complement
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
9
<220>
<221> primer bind
<222> 37267._37285
<223> 99-32281-276.mis
<220>
<221> primer_bind
<222> 37287..37305
<223> 99-32281-276.mis complement
<220>
<221> primer_bind
<222> 37517..37535
<223> 99-32281-26.mis
<220>
<221> primerbind
<222> 37537._37555
<223> 99-32281-26.mis complement
<220>
<221> primer_bind
<222> 39302..39320
<223> 5-298-376.mis
<220>
<221> primer_bind
<222> 39322..39340
<223> 5-298-376.mis complement
<220>
<221> primer_bind
<222> 39670..39688
<223> 99-23460-199.mis
<220>
<221> primer_bind
<222> 39690..39708
<223> 99-23460-199.mis complement
<220>
<221> miscbinding
<222> 6479_.6503
<223> 99-32284-107.probe
<220>
<221> misc_binding
<222> 15222..15246
<223> 99-5602-372.probe
<220>
<221> misc_binding
<222> 15856..15880
<223> 5-290-32.probe
<220>
<221> misc_binding
<222> 16717..16741
<223> 99-22573-321.probe
<220>
<221> misc_binding
<222> 18299..18323
CA 02368672 2001-09-24
WO 00/56924 PCT/IBOO/00404
<223> 99-22586-300.probe
<220>
<221> misc_binding
<222> 18560..18584
<223> 99-22586-39.probe
<220>
<221> misc_binding
<222> 22894..22918
<223> 99-5596-197.probe
<220>
<221> misc_binding
<222> 23163..23187
<223> 5-293-76.probe
<220>
<221> misc_binding
<222> 23241..23265
<223> 5-293-155.probe
<220>
<221> misc_binding
<222> 26094..26118
<223> 5-294-285.probe
<220>
<221> misc_binding
<222> 30452..30476
<223> 99-23454-317.probe
<220>
<221> misc_binding
<222> 30657..30681
<223> 99-23454-105.probe
<220>
<221> misc_binding
<222> 31238..31262
<223> 99-15528-333.probe
<220>
<221> misc_binding
<222> 35136..35160
<223> 99-15798-86.probe
<220>
<221> misc_binding
<222> 36789..36813
<223> 5-297-209.probe
<220>
<221> misc_binding
<222> 37274..37298
<223> 99-32281-276.probe
<220>
<221> misc_binding
<222> 37524..37548
<223> 99-32281-26.probe
<220>
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
11
<221> miscbinding
<222> 39309..39333
<223> 5-298-376.probe
<220>
<221> miscbinding
<222> 39677..39701
<223> 99-23460-199.probe
<220>
<221> misc_feature
~222> 17427
<223> n=a, g, c or t
<400> 1
aaattgattc caggctggac gcggtggctc acgcctgcaa tcccagccct ttgggaggcc 60
aaggcaggtg gatcacctga ggtcaggagt tcgagaccag cgtggccaac atggcgaaac 120
cccatctcta ctaaaaatac aaaaattagc caggcatggt ggcacgcgcc tgtagtgcca 180
gatactcggg aggctgaggc aggagaatcg cttgaacctg ggaggcagag gttgcagtga 240
gccgagatcg cgctactgca ctccagtgtg ggtgacagag cgagactctg tctcaaaaaa 300
aaaaaaaaaa aattgattct agtcaatagg tatttatttt ggggagtaaa gagatgggaa 360
gaattagaga aaggaagagg aaaaacaaaa ataaatagca tgcagataat gagaaaatag 420
actcattttt acagctgtga gctcagacta aaagataaac aatgctatta ctttggaata 480
taattctaat aacacaaaaa agactcacag accacaatat gtatttattt tgtggcaaag 540
gtgtcatttt tagaaagaaa caaatgtgtt caattttgct ttccctgttt ttaatgaatt 600
aagaaaggtc ttctcttacc ttcttttgac tgtcatcatt cctctcatcc aggaatattt 660
acaggtttgc aagaagacac catctttaag tagtttttag gcacttttac aatactgact 720
aaattgtata aaatagtatt ataaattaat atttaaatta taattttatg acgtgaaaat 780
caacaacgaa tgtcaatttc acttcgtttt gactgtcatc tgtggcctct ggagtccctc 840
acttaaatca ttggtccttg gttttatttt ttaagcctat taaaaaggag gatggggccg 900
ggcactgtgg ctcacacctg taatcccagc actttgggag gctgaggtgg acagatcatt 960
tgaggtcagg agttgaagac cggcctggcc aacaccgtga aaccccatct ctactaaata 1020
tacaaaaaaa aaaaaataca acaacaacaa aaaattagac gggtgtggag gcagaggcag 1080
gagaatttct tgaacctggg aggcagaggt tgcagtgagc tgagatggtg ccactgctct 1140
ccagtctggg agacagagcg agaccctgtc tcaaaacaaa caagcaaaca agcaagcaag 1200
caaacaaaca aaaaacaagt tgggcatggt ggctcacccc tgtaatccca gcactttggg 1260
aagccgaggc aggcggatca cctgaggtca ggagtttgag accagcttgg ccaacatggc 1320
gaaaccccat gtctactaaa aatacaaaaa ttagccgggc atcgtggcgc ctgcctataa 1380
ttccagctac tcgggaggct gaggcaggat aaactcttga acccgggatg tggaggttgc 1440
aggttgcacg tgacatagcc gagatcgcgc cattgcactc cagcctgggc aacaaaagcg 1500
aaactccatc tcaaaaaaaa aaaaaaacca aaaaaaaaaa caaaaaaacc agaaaaacaa 1560
aaaaacaaca aacaaaaagg gaggatggta gataactgtc cagatacttt ccagctttgc 1620
cgctatatga actattcctt ttgtttagtt ttcagcatgg gagcttctgg cacttttacg 1680
tactttccag cttctgggga ccggctgcct agaataacag gcatttgccc cagaggccgt 1740
ggagtggcct cactttgggg tcgtgggcag atcgctggct cccacgcctg gacttcggga 1800
tcgcaggcag gatcccttcc agcccaagca ctccgcccag gcgcgccagg cagagccccg 1860
ccccatcccg ccgttccctt cagccccggg gcgcggatct tgcatctgaa actgagcaga 1920
gcagggcgcc gggcagggcc ggcgggccac gtgataagcc cggaaacagc tccgccccct 1980
cgcttcctga gccgccacat cccggcagcc ctcctacctg cgcacgtggt gccgccgctg 2040
ctgcctcccg ctcgccctga acccagtgcc tgcagccatg gctcccggcc agctcggtga 2100
ggccctagcg gagcggcgcg gtctgcgtcc tcgcctgcgg ccccccacgc tcccgccttg 2160
gcggcggccg gcgggacccg gcactgcagg ggcggcgctg cgggattgaa agccagcgtc 2220
cccgctcccc ggccgggccg cagcctgcgt ggggcccgcc ttagagcagc tcgcgggtgt 2280
aagacctggg gaggcccgga ccaggcctgc gaacgcaggg tccaggtgct ggccttgcga 2340
ttcgagaatc tcctccccca gaccctccca aggccttgca gaacacagtg caatgtgctg 2400
cgaatcatga gaaaaaatgt cttctctttc agccttattt agtgtctctg acaaaaccgg 2460
ccttgtggaa tttgcaagaa acctgaccgc tcttggtttg aatctggtcg cttccggagg 2520
gactgcaaaa gctctcaggg atgctggtct ggcagtcagg taaggcatag ctagttccat 2580
cagaaaggag tgtgatcaca ttaaccagga agtattgtat tccaggcacc agcagcaaaa 2640
cgccttattt actcctccca gtagcgctgt gaggttggtg taatacttcc ccactttatg 2700
gggaaaaaaa gtgaggctca gagagattta gcaactttta agaccctgga cggctgggct 2760
aggtggctca cgcatgtaat cccagcactt tgggaggccg aggtgggctg atcacttacg 2820
gtcaagagtt cgagaccagc ctggccaaca tggtgaaacc cgtctctact aaaaatacaa 2880
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
12
aaattagccg gtatggtggc acacgcctgt aatcccagct actgggaagg ctgaggcagg 2940
agaatcgctt gaacctagga ggcggaggtt gtggtgagcc gagatggcac tactgcactc 3000
catcctgggt gacagagaaa gaccctggac aacacactgg acagatttag catcttctgc 3060
acaagtttaa acagttgagt aagggaaaag taggattgga acccgtgctg ttcctgattt 3120
taggacacta gtcttagccc cttccttaaa cagattaact taggtgggcc acttgttatg 3180
caaggaaaac ttcagttact ttgactggtg atttaataaa actcgacatc atgaagattc 3240
caatttacct gccttccctt agaatctctg gtaaccatat gaaggtgcaa atcattcatt 3300
cccacgttca caagcccttc attagctgac taggccatgg aaagaaggga cttagaaatg 3360
attaaccagg atgccattgc tgatgtcaag gagtggcatt ctgggaggag agggaggaag 3420
tctactgaaa ggtagaaaaa tgaaaaaaac tataggatag ctatttaata aacggcagtg 3480
agagttggga ggaacgagat gaccacccct gaaacatatt ttagaatgaa agagatgaag 3540
gatatctgtc tgctttttgt tgccatgtgg aaaatttgct tcttggtttc tagatccatg 3600
tagaggtagg ttattacctt ttctttgtgc agttcccagc tatgtgagca gtacacagct 3660
tccctaagtt taacaagttc agatggtaag aatgccttac tttattaaca aatacataac 3720
tgtatatttt cggatgtctt tttgcgtctt gtctctgtgg ttttcatggg aaacctgtga 3780
gaatcgtggc aggcaagttg gctctttgct catcagcaac cgaatgagaa ttcaaagtcc 3840
agacctgcgt gctttccatt gggccaagtt gggtcctcct atggtaatgg aaccttcgta 3900
aagcaaaatc ctcatctgta gtctcttttt tttccccacc aaactttgta ttatggaaac 3960
atttcagaca caatcaaaag tagaaaagta taatgatccc ttgggtccta tatctgtcac 4020
ccactttagg tacttatcaa ctgattagtc ttgtttctgt acctataccc actcctcact 4080
tatcttcatt atggtcatat cttctttaaa aactggaaat gtaaatgtgt ttctcttcta 4140
tgaaaggata agaagttcac catgagtgat ttatttagac cccagcacag taaagttcac 4200
tgaatgaaat gtaaacaact tggagcaatt gttttttctt caacaaaggc cacattcagt 4260
tgcagtggta ttttattcag ttgcagtggt attttacata tataaactct tatagtcctt 4320
ataacaactt tttagtttag gtagtgttat cctcatttta taggtgagga aactaagcac 4380
aacgtagtta agtaactcgc tcgagattac acagctagtt aagaggccaa gatttttttt 4440
ttttttctct gagacagagt ttcactcttg ttgctcaggc tggagtgcaa tggcactgtc 4500
tcagctcact gcaacctctg cctcccaggt tcaaacgatt ctcctgcctc agcctcccaa 4560
gtagctagga ttacaggcgc ccgccaccac gcctggctaa tttttttgta tttttggtag 4620
agacagagtt tcaccatgtt ggccaggctg atctcgaact cctgacctca agtgatcgcc 4680
cgccttggcc tgctgggatt acaggtgtga gccaccgcac ccgaggccaa gattttgtag 4740
gggtggaaag gtgtgatcct ttgctctcca tcgtaaacgt cacggccaat atttttataa 4800
gagaagacag gttataataa gagaaaagca taacaaattt atttaacaaa gttttacatg 4860
acatgagagc cttcagaatg aagacccaaa gacagaggaa aaaccatcca tttttatgtt 4920
taggttcaac aaagaatgga cagaaaggtg gaagtatgat tggacagcaa ggatatggtg 4980
tatgctagta gactgaggtg gagaaagcca ggaaagcctg tctgtccaga ttcttcttgg 5040
cttctctaaa attctttctc caccccctaa ggatctcctg acctactaat gggcaaggag 5100
atgagaggat ttctttacgt ccagctccta gacagaaagc cagtggaaag ttagaatcat 5160
aagtttaaat cttatgactg gctttgggga aaaagagttt tagtttctgt aaactgccct 5220
ggggaagaga aattctcatt tctgtgactt cagggaagaa tgaagggtga gaggcaagag 5280
ggcaggagaa agctatatat atattttttt tgagagggtt ttactctgtc acccaggctg 5340
gagtgcagtg gcacaatcat ggctaattgc agcctcgatc tcccaggctg aggcaatcct 5400
tccacctcag cctcctgagt atctgggact acaggtgcac accagcatgc ccaactaatt 5460
tttgtatttt tcatagagat ggtttcacca tgttgtctgt ctggtcttga actctagggc 5520
ttaagcaatt ttgcctgcct tggcctccca aagtgctggg attacaagtg tgatccacca 5580
tgcctggaca aggtcttggt tctggctggg cgcagtggct cacccctgta atcccagcac 5640
tttgggaggc tgaggctgct ggatcatctg aggtcaggag tttgagccca gcctggccaa 5700
catggtgcaa ctccatctct gctaaaaata caaaaaagaa tcagccgggt gtggtggcgt 5760
gcacctgtaa tcccagctac tcaggaggct gaggcaggag aattgcttga acccaggagg 5820
tggaggttgc agtgagccga gattccacca ctacactcca gcctggatga cagagagaaa 5880
cactgtctca aaaaaaaaaa aaaagatctt ggttctgagg ctgcttctga gcatattttt 5940
gggtgttgtt atctgcaata attttatccc aggaagttaa ccattacctt gtcatgaggt 6000
aaactgatgg attttaatta gtgagtatac agcagtgagc atacatagaa ttatttcccg 6060
agactacagc cattatgtat aaattcttca gttcaaagga gtagcaagct ttttaattac 6120
cccaaatgtt taattctaga aataaatccg ccttctctaa gttttaagtg actgtcttca 6180
tttggaggaa atggatttta tttcctgaac cccaggaaaa gaaattttga atttgaaaac 6240
acttatgtca ccagtttgtg gatgttggaa ctgtgtgtgt cattggtcaa acaccagtca 6300
tttgcaaatg gctcccctta acaggagaat ctacccagga aattccgtat cttactggca 6360
gaacttgctg ctttaaagtg tttattatag tctcagctac ttggaggatt acttaataag 6420
cccaggagtt ctaggctgcc atggactgta atcttgcctg tgaataatca ctgtactcca 6480
ggctgggcaa yatagtgaga tcccatctca aaaactatat atgttgattg tagaaaattc 6540
gaaaatatgg acaactataa agaaggtagt aaaaatggat tgtaattctt aacatgctag 6600
tatatttttc ttcaagcatt tttataagca tgtactttat ttcatagctt acacagaaga 6660
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
13
tagatgcagt tttgcttcct gctttattta tttttatgta tttatttatt ttttgagacg 6720
gagttttgct gttgttgccc aggttgaaat gcagtggcac aatcttggct cactgcaacc 6780
tccgcctccc gggttcaagc gattctcctg cctcagcctc ccaagtagct gggattatag 6840
gcatgcccca ccatgcctgg ctaattttgt atttttagta gaggcggggt ttcaccatgt 6900
tggtcaggct ggtctggaac tcccttacct caggtgatcc attcgccttg gcctcccaaa 6960
gtgctgggat tacaggtgtg agccacccca cctggcccct gctttatttt ttcttatata 7020
ttgtgggcat ttttacacac cattacaact tataaagtct gccagagtgt tcgtggttat 7080
gactttctag gggtctgctt tgtgatatgg attaatattt actgtccttc acttgaccct 7140
ttgtcacatt gtgtgattat tttttctagt ttactttttt ttcccatgta aaacttttta 7200
tttaacattt ctgtaatcag aactctcaat cttttgttta aaacttggaa agcattcctc 7260
acatatttaa ttttgctaca tgcatgattt tttaaatgct aaacctttga ttcataagga 7320
atatattttt gtttgggttt aacccgttat tccaatagca acaccacttt attaaacagt 7380
cttttatgtc ttcactcact tgatatgcca tcttcatgtg ttgttgttgt tgttttttga 7440
gatggagtct tgctctgtcg cccaggctgg agtgcagtgg catgatctcg gctcactgcg 7500
acctccaact cttgggttca agtgattctc ctgcctcagc ctccccagta gctgggatta 7560
caggtgcccg ccatcacgct cggctaattt tttgtatttt tttagtagag ctggggtttc 7620
atcatgttgg ccaggctggt ctcaaactcc cctcctcagg tgatccacct gcctcagcct 7680
tccaaagtgc tgggtttaca gccatgagcc accgtgcccg gccaaggata tttttaatgc 7740
tttttgttac atactgccaa attctcagtt gtatgtctta gtaatattta atgagtatgg 7800
cttatgattc agtttctaaa tgctctgaaa attataaaac cagtgctgta gtagttacca 7860
attatccctg aacatacaca acagttagga aataaattaa ataaactttt tttcggaagt 7920
aaatagaatt ttacttaaga aataaaatat agtgaaatac tttaaaaaat cagaattttc 7980
ttgttgaatt caggctcaaa atctcttgaa atgaaaacag tagatgcttt gaatagtgaa 8040
aattacaatt cagccacacc agtagtacca ttctgtttat ctgtttttca gagatgtctc 8100
tgagttgacg ggatttcctg aaatgttggg gggacgtgtg aaaactttgc atcctgcagt 8160
ccatgctggt aagtggttgg tatctttaat gtaaaaacag tcagtggttt ccaggaatat 8220
tttagttgat agcgtcctaa aataaaggaa gaaaaaggct caagagaaat ttacatataa 8280
agttaatgtt atgaagttgc tgccagattt cataatacgt tagaactggt ttaaaatcca 8340
gcttgtctta ctacttgatg aattcagatt gttttcctct gcttgctatt agtcctgacc 8400
tgatacctaa tttagagtct ggtgtttcct gctcaagttg ctgaagatat ttagatttca 8460
tcgtatgaaa atacttttaa aatagttcaa acttagaaga aagcatcaca gcgtaactga 8520
cttgcaaagg aatttttttt ttcaaagtgc tttacatttg ttcgttcacc taagaatgaa 8580
ttgtatataa accgaaacgg caagaaactg gtatcctcct agtttgtcag ttgtggtaca 8640
atttggtgaa taaagctgaa tggctacaga tcatcagaca agccattgac ttacagaaac 8700
gcatagactg ttctggaact agcaacagtt ttgtaaaatt ccttttacct tttttacatt 8760
ttattgctca agaaactggg atcaagaact gaagaaaaag atttttaaat atatctctct 8820
tttttttttt tgagacagga ttatactctt gcccaggctg gagtgcagtg gtgcgatcat 8880
ggctcgctgc agcctctgtc tgcttcccag gctcaagcag ttctcccacc tcagcctccc 8940
aagtagctgg gactataggc atgtgccacc acacccagct aatttttgta ttttttatag 9000
agacggggtc tcaccttgtt gcccaggctg gtctcgaaca cctgggctca agcgatcccc 9060
caccttagcc tcccaaagtg ctggtattac agacatgagc cactgtggcc agccagatat 9120
atctgttaat cctaattttt ttgtttgata actccccaac tacatgtttg atattcttta 9180
attaagaata ttatgctggg catggtggct cttgcctgta gtcccagcac ttggagagct 9240
gagacaggag aaccgcttga gcccaggagt ttaagatcac cctgggcaag atggcaacac 9300
cccccttctc tttaaaaaat tgaaaagacc agctgggtgt ggtgatgcat tcctgtagtc 9360
ccagctactt gggaggctga ggtaggagga tcacttgagc cctggaggtc agggctatag 9420
tgagttgtga ttacgccact acaatccagc ctgggcgata gagtgagacc atctcaaaaa 9480
aataaatttt ttttttaatc aatgggattt aatttgattg aagacactat gttgaaagac 9540
attccttaat ctgacttgtt ttttgaagct aatgactttg tttaactttt ttaaattagg 9600
aatcctagct cgtaatattc cagaagataa tgctgacatg gccagacttg atttcaatct 9660
tataaggtaa aaacctgaaa ttaaactttt aacgcattac gaaccaacga caaagactat 9720
gccaaacctg gtgtccctgt gttttcttac tcactataaa cctttactgc gtaccttctg 9780
tgtgactttg tatgtgtgta agcattttgg tttggccaga tttatatacc aaaatacata 9840
ctgaagtttt ttaggaagtt acaatctaaa tcttagtatg tataggttga gtatccctta 9900
tctgaaatgc ttgggaccca gaagtgtctt ggatttcaga tttcttcaga ttttggaata 9960
tttgcaggta acatgccagt tgagcgtccc tcagaaatcc gaaatgcttc agtgagcatt 10020
tcctccaagt gtcatgttga cgctcaaaaa gtttcagatt ttggagcatt tcagatttca 10080
ggttttcata ttaggaatta tcaacttgca caactaactg agttatttgc ataaagatac 10140
tggctgtttc tcttaaatat acgtaacagc tttattgaga tctaatccac ataccataca 10200
actcaccaat ttaaaatgta caaatcagtg gttcacagaa gttgtgcaac cattactgtg 10260
ttagtctgtt ctgcattgct ataaaggaat actagaagct gggtaattta tgaaaatagg 10320
tttattttgg ctcgtgattc tatagacagt acaagaagtg tggtgccagc atcaacttct 10380
ggtgagggcc tcaggaagtt tataatcaca gtggaaagca gagggggagc tggcatatcc 10440
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
14
catgagagaa gaagcaagag agagggagag gaggagttgc ccagctcttt tactttttaa 10500
cttttattct taatttaatt taattttatt ttgagacagg gtctcgctct gttgctcagg 10560
ttggagttca gtggcatgac cttggctcac tgcaacctct gcctcctgtg ttcaagtgat 10620
tctcctgcct cagcctcctg agtagctgag attacaggcg tttgtcacca cgcccaactc 10680
atttttacta tttttagtag agatggggtt tcaccatgtt ggtcaggctg ctcttggaac 10740
tcctgacctc aaatgatgca cccaccccgg cctcccaaag tgctgggatt aggtgtgagc 10800
caccacgccc ggcctgcccg gctctgttaa acaaccagct ctacatgaac tcagagtgag 10860
gactcattat ggggagggca ccaagccatt cataagggat ctcccccgtg acccaatcat 10920
ctcccaccag gccccacctc caacattggg gatcacattg caaaatgaga tttggagagg 10980
acacacatcc aaaccatatt aattgccaca tccaatatta aaacatattc atcaccccca 11040
ccctaaaccc tatacccata cgcatttatt ctccatttcc ccaacgtcct ccagcctcgg 11100
caaccaccaa ttgttacgtg tctgatttgc ctgtagtgga cattttcata taaatagaat 11160
ctaacaatat atggtttttt tgttcctggc ttctttcact tagcatgttt tcaaggttta 11220
tccatgttat agcatagtat caatagttca tttcgttttt agtgctgaaa aataatccat 11280
tgtgtggtca taccctgttt tgtttatcag ttcatttgtt gatggacatt tgggttgttt 11340
ctactttttg aatattatga ataatgcagc tataaatatt tgtgtataag tttttgtgtg 11400
gacatacaca ttcgtttcat tggggtatat acctaggagt ggaattcctt ggtcatatgg 11460
taactatgtt tagcttttga ggaactgcga ccctgtattt cagagtgctg caccatttta 11520
catttccagc agcagtgtgc tggatggggc tccagtttct ccacatcctc atcaacgtta 11580
ctatctgtct ttttgattct agtcattcta ggggttctga agtggcatct cattgtggtt 11640
ataatttgct ttccaaataa tgtggaacac cgttggatgt gcttcctagc cagttgttta 11700
cctcctttgg agaaatgtct gttgagacct cttgtccatt tttagttgag gtatttatct 11760
gtttattatt gagttgtaag tttatttcct ctcattctat ggattgtgtt agcctttctt 11820
gatggtttcc tttgatcatc acaagttttt tctttttttt gagacggagt cttactctcg 11880
cccaggctgg agcacagtgg cgtcatcttg gctcactgca acctccacct cccggattca 11940
agcgattctg cctcagtctc atgagtagct ggggttacag gtgcccgcca ccacacttgg 12000
ctaatttttt tggattttta atagagatag ggtttcacta tgttggccag gttggtcttg 12060
aattcctgac ctcgggttat ctgcccgcct tggcctccca aagtgctggg attacaggct 12120
tgagccacca tgcccggccc acaaaagttt ttaattttga tgatgttgaa tttatttttt 12180
cttttgttgc ttgtgttaat ggtgtcatgt ctaagaaacc attgcctaat cctcagtgat 12240
gaagattttt gtgtatattt tctttctttt tttttttttt tttgagatgg agtttcgctc 12300
ttgttgccca ggctggagtg cagtggcgtg atctcggctc actgcaactt tcgcctcctg 12360
ggttcaagcg attctcatgc ctcagcctcg caagtagctg tgattacagg tgcccgccac 12420
cacgcccagc taattttttt gtgtttttag tagagacggg gtttctccat gttggccagg 12480
ctggtcttga actcctgacc tcaggtgatc cacctgcctc ggtctcccaa agtgctggga 12540
ttacaagtgt gagccaccgc acccggcgtg tgtacatttc ttttaagagt tattttagtg 12600
ttagctctta tacttaagtc tttggttcat tttaagttaa ttttcatata cagacatgaa 12660
atagaagtct tattttattt tgtatgtggc tgtctagttg tctcagcatc atttgttgaa 12720
aagactgttc tttgcccaat cgaatggtct tggcaccctt ggtccatgtg cctgctgtta 12780
tgccagtact acactattac tataggctgg taacaaattt tgaaatcagc aagcgtgagt 12840
cctccaactt catttttctg ttttttgttt gtttgtttgt tttgttttgt tttgtttttg 12900
agacggagtc tggctctgtt gcccaggctg gagtgcagtg gcgcgatctt ggctcactgc 12960
aagctctgcc tcccgggttc acgcccttct cctgcctcag cctcccgagt agctgggacc 13020
ataggcgccc gctaccatgc gcagctaatt tttttgtatt tttagtagag acagggtttc 13080
accgtgttcg ccaggatggt ctcgatctcc tgacctcgtg atccgcccgc ctcggcctcc 13140
caaagtgcta ggattacagg cgtgagccac tgcgcccggc cccaacttcg ttttttttat 13200
tcaagatgac tttgactctt cagagtccct tgcatttcca catgattttt agaatcagct 13260
tgtccatttc tgcaaaacaa cgcagttgga attttcatag gtattgtgtt gaatctgtag 13320
atcaatttgg gaaatgttac catcctaaga atattaaatc ttccagtctg tgaacatggg 13380
gtgtctttaa tttctttaac attgtcttgt gaagtagaat tgttttctta atttttttct 13440
cattcactgc taatgtatat tttaatacaa ttgattttta tatattgatc atatatcctg 13500
cggctttgct gaactcattt attagtgcta aaaggttatt tttgtgaatt tgggattttc 13560
tgtatactag gtggtatcat ctgcaaatag atataacttc actgctttct ttccagtcct 13620
gatgcctttt attttctttt ctttgctaat tgctctggct agaacttcta gtacagtgtt 13680
cagtagaagt ggtaagaatg gacattattg ttttgtttct tttttttaag atggcgtttt 13740
gctcttgttg cccaaggcgg agtgcagttg cgtgatcttg gctcactgtg acctccgcct 13800
cccgggttta agtgattatc ctgcttcagc ctggggttac aggcatgtgc caccttgcct 13860
ggctaatttt gtatttttag tagagatggg gtttctccat gttggtcagg ctggtcttga 13920
acttccaacc tcagttgatc catcctcctg agcctcccaa agtgctggga ttgcaggcgt 13980
gcgccacctt gcccggctaa tttttgtatt tttagtagag atggggtttt accatattgg 14040
ccacgctggt ctcgaacttc tgacctcagg tgatctgcct ggctcagcct cccaaagtgc 14100
tgggattata ggcatgagcc actgcgcctg gcctcttgtc ttgtttctga gttgagtaga 14160
caagtatttt agtctttcac catcaagtat gatgttagct gtgggttttt cttacacttt 14220
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
gtcatgttca gtaagttccc ttctattttt aatttgctga gtgtttctat atgaatacct 14280
gttgaattgt gtcagatcct ttttgtacat ctattgagat gatcaggtgg tttttgcatt 14340
tttctggatt cagtttgtta gtgttttgtt gagagttttt gtgtgaagat acctaagaga 14400
tactggtctc tagttttctt gtgacatttg tctggtttta gtaggagggc agtagactta 14460
ataaagatga gctgcaaaat gtttcccctc caattctgtt cttctgtttt tgtttttgtt 14520
ttttttaatt agttttcagc agttaggctt gtttggagcc tgcccgtaga gctcctcgca 14580
ctacaggcct aggagtggaa ctgtacttca ctgattgtta attttagatt acttctgtat 14640
tttaaattat tcttttggca ttcctgttac catcattttt atgacctctc tgaaggcaaa 14700
acaaatgttt cacccttaga atgctctgat atttttcatc attgtgccaa tccactggaa 14760
aaagaatcta aattctaatg ttctggataa tagtgatcac attccaaaat gagaatgtta 14820
tctgtaatct tgtactttat acttctatta aaatgttcta taaatttttc atggcttggt 14880
g'gttctgggt agctaaggtt atgcaagcag cagcgttgca gtgtgacgtg gagggagtac 14940
tgtgtattca gatccgggga gcacctgctg actaaattac ctctgctcga ccccagcagg 15000
acaccctgac tttttaacac actcgttaga attctaaagt gtcaggctca cagtttatgt 15060
attagttctc tatggctttt gtttacgtta atagtactga cttgtttttt tcctagatag 15120
ctgtaaacca catgagtgga ctttttaatg acagccagat tcgtctttgt tttatagagt 15180
tgttgcctgc aatctctatc cctttgtaaa gacagtggct tctccaggtg taastgttga 15240
ggaggctgtg gagcaaattg acattggtaa gtcagaaaaa ccattttaga agactgagag 15300
gagaggatta tttaaatttt agtgagattt cattttgaat tttattactg aggaaataga 15360
aagaaaatag ctacttctgg attgtgtttt gggattacta taattcattt atattttctt 15420
tttttcattt atattttcta agcttttttt gtatgcagag atgcatctgg gttattttcc 15480
tgatttattt atttatttat tttattttta tttttttgag atgaagtctt gctgtgtcac 15540
ccaggctgga gtacagtggc acagttttgg ctgactgcaa cctctgcctc ctgggctcaa 15600
gtgattctct tgcctcagcc tcctgagtag ctgggattac aggtgcacac caccacgcct 15660
ggctaatttt ttttgtattt tttatagaga tggaatttcg ccatgttggc cagagtggtc 15720
tcgaactcct gacctcaggt gatccacctg cctcagcctc ccaaagtgct gggattccag 15780
gcatgagcca ctgcgcccag cttattttcc tgatttttaa gtcaggaatt aaaatggaag 15840
tacatgcaca atgactttat ttaaaagygg ttcataagct gatagggaca tacaaaaatc 15900
aatacgtctt ttgttcttca aaggtggagt aaccttactg agagctgcag ccaaaaacca 15960
cgctcgagtg acagtggtgt gtgaaccaga ggactatgtg gtggtgtcca cggagatgca 16020
gagctccgag agtaaggaca cctccttgga gactagacgc cagttagcct tgaaqgtggg 16080
atgcactttc atgatattgt aagttacatc catggagtgc agtgtttgcc agaccaagca 16140
gtattcagtt cttggtagat tgcattacct accaaagctt tgcttggagc tgctatcctt 16200
ttgttaaaaa tggagaaacc agctattaca gaggtgttct gtcaaaaaga ttgaaagaga 16260
gctgggcgcg gtggctcacg cctgtaatcc tagcactttg ggaggccgag gcgggcggat 16320
cacctgaggt caggagtttg agaccagcct ggccaatgtg gtgaaaccct gtctctactg 16380
aaaatacaaa aattagccga gtgtggtgtg gtgtggtacg cctgtaatcc cagctactcg 16440
ggagactgag gcaggagaat cgcttgaacc caggaggtgg aagttgcagt aagctgacat 16500
cacaccactg cactctagcc tgggcagcag agtgagactt tgtctcaaaa aaaaaaaaaa 16560
agattgaaaa agaattggaa aggggtaatt tgttgggcca ttcagtgctt tgctgtcatt 16620
catggtgtca tcgtgttgtg cagccaccac atagcactga atagtgagga gatgttgttt 16680
gctgtggacg tagaaaacca tcttgtcccc actttggaaa gtgctgctyg tgtctcacaa 16740
aacttatgct ttttgtaggc attcactcat acggcacaat atgatgaagc aatttcagat 16800
tatttcagga aacagtacag caaaggcgta tctcagatgc ccttgagata tggaatgaac 16860
ccacatcaga cccctgccca gctgtacaca ctgcagccca agcttcccat cacaggtaaa 16920
gcccgagcgt tctgtggcat ggtttgctgt gcctggagag tgtgtgtttc tctgtattgc 16980
atctgatgtg gttcacattc agaagatgat acattccgta atgcctcctg tagccatctg 17040
ataaccatct ggtttgagac gccatttgga ctgaagagca cagaagttga gtagtctgga 17100
gttctgaaaa gatgtcatgg gggagatgga gattggaatg tctctgccac gtagatcttt 17160
ttgaagacat gagacaggat gggatggcct ggattttata gagaagaggg tcaagaaagc 17220
cttggggtaa aggcttggta ggtgtactgt ggaggaacca gcagaagaaa agttgtgaaa 17280
ttccaacctt ttagaatgca tttgagaaca gaaaagggac tgtttgtttt gcacatattg 17340
taagaagccg ttgccttgga atattggatc tagaagccca attgaagtag gccaaagcag 17400
aaatgtgatg ttaagaacta acaacgnata agaggtttct tggaacaaga aagtaggact 17460
ttctctgggt ttgtagagtt tagagagggt ttttaaatgt gtgtcctgtt ctgttccaaa 17520
ggtaggctgt gagagcctgt cctgctctgg tacaagggag agtaggggca gggacagagg 17580
tgagagtccc aggcatggag gccttggaat cccaagcact accggtgcag ctggccttgg 17640
ctgagagcaa gggcagcctc cttcctgcct ggggcggtca gcatttggtg agggggtgca 17700
gcctgggctt cctttccctg tcatcagctg agagagcgat gggggagggg agcagcgtgc 17760
caggtgggag gagaatctga aagtttcttc acgtttgccg cgttctattg ttctgcctca 17820
gatagatttt tttttttttt ttaaattaag acaacggagt ctcgctctgt cacccaggct 17880
ggagtgcagt ggcgcaatct ctggttcaag cgattctcct gcctcagcct cctgagtagc 17940
tgggattaca ggtgcgcacc acaatgcctg gctagttttt ctgtttttag tagagatggg 18000
CA 02368672 2001-09-24
WO 00/56924 PCT/IBOO/00404
16
gtttcaccat gttggccagg ctggtctcaa actcctgacc tcaagttatc tgcctatctc 18060
agcctcccag agtgctggga ttacaggcgt gagccaccac acctggcctc agatctcaga 18120
tacactttga cactaacact tcccaaatcc ccacagacac cctgtgaagt cctgtaggct 18180
gaagtaatct aagtaatttg ttttctctct cctttctggg cttcacagca cagtaatctt 18240
ttaaaaatgg aaatcggatg atgtcagacc ctacttaaag ccattccttg gcttttctta 18300
agcactttgc sctgccccac aaaccctgtg ctgcctgcct gcctcctatc tgccccctca 18360
gcagctctcc ctgcgcctgc tgctagcccc tccgccctgt gcctccaagt gggcccagct 18420
ctttccactc cagcgcctct ggctccttgt ccccttcacc cagaattctt ccctctgccc 18480
ttcaccgtcc tggtttcttg ttattcaggc ctcacctcct cagagaagcc ttccttgacc 18540
gcttagacca aagtagcagc ccatcatgct tycatcccat caccctgttt tgctgcatgt 18600
gtttctattc aatctgttca aattgtcttc ccccattagg atggcagctt cctttttttt 18660
'tttttttttg agatagagtt tcactctttt cgcccaggct ggagtgcagt ggtgcgatct 18720
tggctcgctg caaactccac ctcccgggtt gaagtgattc tcctgcctcg gcctctcaag 18780
tagctgggac tacaggcgcc tgccaccaca cctggctaat tttgtatttt tagtagagat 18840
agggtttcac cacgttggcc aggctggttt tgaactcctg acctcaggtg atctgcctgc 18900
ctcggcctcc cagagtgctg ggattacagg catgagccac agcgcccagc caggatgtca 18960
gcttttgaga gcagaagctg catctgtttt tgtttgtcgc tgaatcccca gggcttagaa 19020
gactgcttgg cacattccct aaatgtttac tgaatgaata attagcaacc tgacattttg 19080
cacaaatttt tccttgtagg acttcttgaa actgatttgt ctgctgttat atctgaccta 19140
tttcctgctt atagtttata ttctccatta agattctgag acaaaaaaat ccccatgtac 19200
tgtcagttct tatacttttc attttccaaa gcattttcat tggactatat aaaagccttc 19260
attgttcctt agactgtgtg tgcgtttctt ttgaagttta aagtattatt tgtggtaaac 19320
atggcagagg cggccctgga cacgtaaata agcgtttctt gtgcgagatt cccaggattt 19380
ctccccaaca tgggcttatt tccacaatag aaaaggcact ttgccttttc atgtggtttg 19440
agtatttggc agtgatgtta ttacagagtg tatggttgcg attgtatcta aaacaaaatt 19500
gaagcaagaa caaagatggg gttgtgtgag tgtgtgtatg tgtctgagat tttaatgact 19560
ggtttgctta tgattggaaa gaagaaaatt cctatttatt tcagctattt actagattaa 19620
gcaactttca gcatttctct ggcactttct atttcagtta ttaatgtctt tgaaaatttg 19680
atatttgaag gggaaccgga tactcttttt tttttgagtc agagtcttgc tctgtcgtcc 19740
aggctggagt gcagtggcat gatctcggct caccacaacc tccgcctccc aggttcaagt 19800
gatctcctgc ctcagcctcc tgagtagttg ggattacagg catgtaccac caggcccagc 19860
taatttttgt atttttagta aagacagagt ttcaccatat tggccagacc agtctgaaac 19920
tcccaacctc aggtgatcca cccaccttgg cctcccaaag tgttggggat tacaggcatg 19980
agccaccgca cccatccaga tacgcttaac tatgagatgt ttcagaaacc aaaagttttc 20040
tcccacttat caaccttagc ctaaaccatc tatgaggagt gggagggaga tgggggagtt 20100
gtacatatgg tgttttgttt tattatgcct gttgtatgcc ttaggggata ttttacttct 20160
attgtttact atttattgcc tagtttgttt tcttctgtgc ctctcgcata aatttcatga 20220
ggacagtgat tttatttgac ttgatttttg gttgggggaa ttattttttc aagttctttt 20280
taaaacgggc tttatttttt tagagtagtt acaggtttac agaaaaaata cacggtgatt 20340
atagggaatt tccatatacc ccgcttcccc gcagtttctc ctattaacat catgcattag 20400
tgtggtgtat ttgttacaac tgatgaaccg attttgattc attattaacc aaggttcata 20460
gtttaacatt cattctttgt gttgtacatt ctgtgtattt ggaaaaatgc acagtggcat 20520
gtctccccca ttacagtatc atgcagaata gtttcaatgc cctaaaactc ccttgtgctc 20580
ctccgcctca tccctccttt cccttctccc caaccctttg caaccactgt tatctttttc 20640
tcttttcttt tttttttttt tggaatggag tcttgctatg ttgtctgagc tggttttgat 20700
atcctgggac tcaagcagtc cttccgcctt ggcctctcaa gtagctggga ttacaggcat 20760
gcaccaccat gcccagctgt ttttactgac tctgtaccca tttccagaat gtcagatagt 20820
tgggatcata cagtatggag cacttttagg cactttcagt taacaacatg cattaagttt 20880
cttccacatc tttttgtggg ttggtagctt atttcttaat gctgaataat atttcactgt 20940
gtatgaatgt accacagttt atccatggac atattgaagg gcatcaaaac ttcaaggaca 21000
gtttttggta gtttatgaac aaaattgcta taatcatttg tgtgcaagtt ttcaactcat 21060
tttgataaat acctaatatt ttcttttatt gtatactatt tattgcctat tttttatttg 21120
cctccccagt aagctccata aggacagtga ctttttttta agagggttcc agggccggtt 21180
tttttcactg atgtatcctg aatacctaga acagtgctta acatcgtagt aggcaactca 21240
gtaaatattt ggcaagttct ttaaaaacta tcttaaccca tgaaggacat gaaattacac 21300
atttttgttt tttgggttgt tttttttttt ttttttgaga cggagtctcg ctcttgttgg 21360
ccaggctgga gtgcagtggc gcggtttcgg ctcactgcaa cctccacctt ccaggttcaa 21420
gcgattctcc tgcctcagcc tccctagtag ctgggattac atgcgcctgc caccacaccc 21480
agctaatttt tgtgttttta gtagagacgg gggtttcacc atgttgacca ggctgatctc 21540
gaactcctga ccttgtgatc cacccacctc ggcctcccaa agtgctgggg tcatgggggt 21600
gagccattgt acccggctca tttttgtttt taattagtgc tggcccacca tttctttctg 21660
tgtgctgaac ttcttatgtt tattcacaag agatactagc tcctaaaaaa atctgccagt 21720
gttgagaaac taggtaatgg aaaactctga agttgcagtc tccttttaac tacagttctg 21780
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
17
ctttaggcag agtctgttga gtttgccatc aaagaagaaa gaaaaaacac attatttctt 21840
ctcttcttcc acccacagct gttgctttag cttgtaatat aatttcttac atcgataggg 21900
tttataactt taattttgta ctgtctctgt gattacctca attgtttaat cttagttctg 21960
tatttacatg aattcctgac tgatcactag tcttgtcata gtttctccca tccctaaatc 22020
tcttattgtg gatttatttc tagactggct ggttctggtc ctccagtcgt gtgtgtgtgt 22080
gtgtgtgtgt gtgtgtgtgt gttcatgtac atgtgcattt tttttaaaga tgggctcagg 22140
ctcagttgct ttgttcttta tcatcaagat tttatgtttg tgtgtatctg tttggctctt 22200
aattatactt aaaacatttg tttagtcatg ttattttttt gagaagtgtg catacatctg 22260
accttactga tcctgcttag taatgtgcat gtatattttt acatttagtt ctaaatggag 22320
cccctggatt tataaacttg tgcgatgctt tgaacgcctg gcagctggtg aaggaactca 22380
aggaggcttt aggtattcca gccgctgcct ctttcaaaca tgtcagccca gcaggtaaag 22440
ctctgtgctc tggaaagctc cagaattgtt cgaaaggcat ttcttcttaa atttttttga 22500
atattaacaa gttctaatgt gaatatagac atgctatgaa atctgaaagt catctgttga 22560
taacagtatc aatgtaataa tataaaatct gatacacaca cacacacaga attgtgtaat 22620
attgtcagga aaatgaggca ggaatttgtt tgtggggtgg aaccataaaa gtttagaaaa 22680
taaaagtgaa atgtcctaat gcaatagact aagtcttgtg ccacatcttt gaaaatgtaa 22740
tggagtaaac ataaagaagt tcttctttgg actctttatc tgaaacttct tatagtcgtt 22800
gtatgagata ctttgcttct gtgtttaaat catgaactcc gcaattgagt tgtctgtaca 22860
ccacatcttg ctgacagtcc tccagcacat gatttgttct caggtrttta ttgggtgtta 22920
aagtgatgtg agtggggaag aaataaatga tgtgtgatat caaagtgata tcaagcagaa 22980
catagagaaa gagtgagtca gtaatgaagt aaacgagctt cacctacacg ctgggtactg 23040
tcaggcttca gtggctctgt ggttttggag ggcagagagg agatttccct cacactgctc 23100
ccacaaccat atttttagtg tataattatc tggctaaata gatttctgtt taatttagca 23160
caggcactag aaaaygtgct gcgtagactg ggtgttaaga aaactgtctt gatttaggag 23220
ttgacaggta gtaactttag ctttctttgt ttrtgatttt actattttct aaccaggtgc 23280
tgctgttgga attccactca gtgaagatga ggccaaagtc tgcatggttt atgatctcta 23340
taaaaccctc acacccatct cagcggcata tgcaagagca agaggtcaga ctcatagggc 23400
tttttgattt gggggagaaa gaaaaagcaa tattttatcc taaatagaat aaagaggata 23460
gaataaagaa aaaatatata gatattctgt ataatatata gatgaaatta aggacttcta 23520
tctctatgta aatatacagt tattctatat aatatagttt gattaaagta aaataccctt 23580
tgtttcatta taggatttcc tatttaattt gttttattaa tatttgagat tgattctgtg 23640
agtacctcaa aatacatttc tttataaaag tgtatgttag tattttaggc cgggcgcagt 23700
gtctcacgcc tgtagtacca ggactttggg aggctgaggc aggcagatca cgaggtcagg 23760
agatcgagac catcctggct aacacggtga aaccccgtct ctactaaaaa tacaaaaaat 23820
tagctgggcg tgatgggggg tccctgtagt cccaggtact caggaggctg aggcaggaga 23880
atggtgtgaa cccggcaggc ggaacttgca gtgagccaag atagcgccag tgcactccag 23940
cctgggtgac agagcgatac tccgtctcaa aaaaaaaaaa aagtatttgt tagtatttta 24000
gtgtaataaa tggaaacaat tggaatttgg tcaatgtaat tttgctaatt tagcctattg 24060
aatgcatttg ttattgttta atatgtgacc aaaattgaaa ttacaaaaag ctattctttt 24120
tttttttttt tttgagacag cttcttgctc tgttgtccag gctggagtgc agtggcgcca 24180
tctcagctca ctgcaacctc cacctcctgg gttcaagcga ttctcctgcc tcagcgtcct 24240
gagtagctgg gattacaggt gcgtgccacc acacctggct aatttttttg tatttttagt 24300
agagacatgg tttcaccatg ttggccaggc tggtcttgag ctcctgacct caagtgattc 24360
acctgcctca gcctcccaga gggctgggat tacaggcgtg atttatggct cttctaactt 24420
tactacattt ggttagagtc atgttttatc agagacattt acttgttgaa tttaataacc 24480
catgaatatt cctttaatat ttctgaaagt tcttgagaaa ctgttaccat agtttatcat 24540
ctacagcagt ggtcagcaac ttcagctgaa tccagcctgc caccctgttt ttgtgaatat 24600
agtatatata aattggtcct tcacagaaaa agttttctga gtgtcactct agagggtaaa 24660
attaaagaat actttcttac tggttacttc agttaaatga cttattgatc tgttaaaatc 24720
ttacgtaaac agtaagatta gctttagaga aatgaatttt atatgacagt ggagaatttt 24780
attacttttt ctgtatcagg atactttgtg ataaatacct cattttaatt ttatttacag 24840
gggctgatag gatgtcttca tttggtgatt ttgttgcatt gtccgatgtt tgtgatgtac 24900
caactgcaaa aattatttcc agagaagtta gtggacattc atgtatctta atctgtgtgt 24960
tgaataaagc tttatttgtt cataatgtta attgaaacca taacctataa tatttatatt 25020
tataatatct tttaattagc taagtttatc atttaatgtt catattttaa atagaacatg 25080
aatcttaaat tgttaatttt gaaatgtagt gaattattgt ttcagatgga tatttggctg 25140
ttatgatgtt ttttacttat atatttattt taaattcttt gttgtcttca tgccaaagtt 25200
attgtttctg aagagcagaa gttgtaattt taggtggtga tgcgatataa tccatcttac 25260
tgcatagtgg gtaaaaaaca gaaactacat taataaactt accccatcag ttgaatacca 25320
aacaggtatt ccagacaggt gggggtactg ttggtagtag gaagacagga agaaaagaga 25380
aaaagattaa atagctcaga aaggggaaaa aaataagcag aggtgaaggc tgaaggagca 25440
tgtggcttgg cactattttg ttgctttagg attctttcca tggccactgc catccctggg 25500
ttacaggtta caggagggct gtgacatttg tatctttcgt catgtcaaat ctctgattta 25560
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
18
atctggtctt gctgttaagg aaaacgcctt gccggaagag gtcatttaac catgtactag 25620
gagagtcagg actagaatgt gctcttgctc attcaaaaca ctccctgaag aaatatttcc 25680
tcctgaggta catgtgttaa gagcaaatca gtcatatttt cttagtttaa actgaaacga 25740
acaaaaactt ggagaggaca gaaagttagg atctgctata cagaaagctg tattctaata 25800
gatttctctt ttcaagtata gcgttagcat tgtttgttgg aggcagaaac cagaatatgc 25860
tgccacatta tttaagactg ataattgctg ctagattttt ttaagaggtg ttctctgatt 25920
tttaaaaata gaaattaaaa tttaatattt ttgcaggtat ctgatggtat aattgcccca 25980
ggatatgaag aagaagcctt gacaatactt tccaaaaaga aaaatggaaa ctattgtgtc 26040
cttcaggtga gtgcaattca tgtttgaagc ggtaatttgc tcttttattc tgtgtctctt 26100
tctccsttgt ttactctttc ctttatactc atacccttct agtttatcct tattatagtt 26160
tattttcccc aatcctgcat ttaaaaaaat actattaact tggggttttg agatgagagt 26220
agctttctta ccctcaattt taactttttg ctggttgtga tggcttatgc ctataatttc 26280
agccacttga gaggctgatg tgggaggatg gcttgaggcc aagaattgga ggctgtattg 26340
tggtactatg atgcctgtga ataacgactg cactccagcc ttggcagtgt agtaagacca 26400
tgtctctaaa atactaataa tagtaacaat atttaatgga aagtctctcc atccttctgt 26460
taccttttga aaagataact attttaaatt tcttgcttat ctttcctgaa atagtctata 26520
tattcagcaa atatttgcat gtgcatttct ttttctattt tcctattgac acaagtgcac 26580
cctgcttcac cttcttggaa gtttgtcctg tatctgtgta ttttaactta cctagacagt 26640
caccaatctt tagctactac aaatacccct gcagtgaata acctagaaaa gtcctgagag 26700
tgaaattaag gcttaataac aagctctatg tatattggta atcttaattg atactgcccc 26760
ttttagatgt tgtaggcaaa ttcattgtcc taactgaaat atataaggat gcctgtttcc 26820
caacactttt gtcagcattg tggttttata cttttaaaaa gtctctgcag ttagaaaatg 26880
gcatctcaat gtagttttaa cattgtgtgt ggtatgagca tcttttcatg tttgaatgtt 26940
tttattcctt tctatgaact gtctttccat attctttgat gatttttcta ctaagttgtt 27000
gttggctttt tttttttttt aactgatttt tatgagctct tctgtattgg gaaaagtagc 27060
tcatggtcta agattatgca ttgataaagc aaagagttat ttcaatttaa agtaaaaatt 27120
cctaatgcta cttttcatag tattgatatc agaactctgt ggaatcctaa ttccttacaa 27180
ggaaacttat taagtatatt cttttgattg taagatacca ataattttat agttcaccat 27240
agattccatg tcagctattt aaggaaagaa gagaatacca cattaaagtt aatacttatt 27300
tttttttagc cacttgacag tctgttgccc aggctagagt gcagtggtgt gatcttggct 27360
cactgcaacc tctgcctcct gagttcaagc gattttcctg cctcaccctc ccaagtacct 27420
gggactacag gcatgtacta ccaggcctgg ctgagttttg tatttttagt agagacaggg 27480
tttcaccatg ttggccaggc tggtctcaaa ctcctgacct caagggatct gcccgcctcg 27540
gcctcccaaa gtgttgggat tacaggcgtg agccaccgcg cctggccccc agtttcttaa 27600
tcaagttttt ttgttgttgt tgttgttaag agtgttatgt acttaacaac cattagcatt 27660
ttcttgtagc atcatggaaa atattttcca aaacctgagt cattttcttg atctctttaa 27720
cacctgatag ttggagagtg tcaaaggaat atcaggactt cattggtatt ttacttattt 27780
gagaatttgg cttagtttgc acattggacg catgattata agcatgagac aacttatccc 27840
tctacctgac taatgataag tttctcctag catcagttaa taggagagca tatcaatttt 27900
agaaatttta tagtgcataa tacaaaggaa cattttagaa ttcaagaaaa tgtagattaa 27960
acagaactaa tatttacggg gtcttgctta taaattatat gtcagggctt tctaaatcaa 28020
agatcatcca aagcataaag aagaaatagt gtcaagttta aaaaaatatc cagggagaga 28080
gtagtgttac acgtattgtt ccacacacct taatgttctt tcggctcaaa atttgggaga 28140
ggaagatggc agtaaatgat acaaattcct tccaaatagc aaagacacag attacaaatc 28200
ctgttttctc acttttagct ttcagttcct gggttagctg ggtattttgt aacagatttt 28260
tagaaaggaa aattagaatt taatttttgt gccattgctt atttaattag cacatctgtt 28320
ttatctgctg accttccaaa agaaccatat aggtagtaac ttaaccaaaa atacactcac 28380
tggaatcatt gttttttaac atgttgtata taggttgtaa aatctataaa atactattgg 28440
gtagtaaaat ctataaaaga ctttggagaa aaatattagc gcctggctgt tgtgtcatac 28500
cagaatgttg aattgtatca taccagaatg atttaacaca tcatttgttg tgtgagaaaa 28560
attagaaaat tagaaaactg taaaaaatta gaaacatgca tataacttta cttacaatga 28620
aatcttttgt atataaatta aatgaaaaat ttgagggaag tggagagatt aactttaact 28680
tttaaaattt gtattttaga tggaccaatc ttacaaacca gatgaaaatg aagttcgaac 28740
tctctttggt cttcatttaa gccagaagag aaataatggt gtcgtcgaca agtcattatt 28800
tagcaatgtt gttaccaaaa ataaagatgt aagttgggaa gtatctgaac tgactgctag 28860
taacttccat tggtctgttt tcaatactta atgagcttta catttattaa ggtctggatt 28920
tggaactggt ctgtatgcac acggctagct agcttatctt tgagttgtca cataagcttt 28980
ggagtttaag aaatgaatca aaggccatgt tagaagcttc tgtgcatatg tacacactag 29040
agataagtag ctttaattgt gtataaatgg gatctgtctt ccataatctg gctcctgtaa 29100
cttggctttt ttccatgtgc taggctacaa atgtgttttt atatcaatac atatagatgc 29160
tgggtgcagt agctgacatt tgtaatccca acactttggt aggccaaggg gcagctctct 29220
tgaggtcagg agtttaagac cagcctgacc atgtagtgaa accttgtctt tagaaaaagt 29280
acaaaaatta gcctggcgtg gtagctgcgc ctgtaggtcc agctactcga gaggctgagg 29340
CA 02368672 2001-09-24
WO 00/56924 PCT/TB00/00404
19
tgagaggatt gcatgagtct gggagatcaa ggctgcagtg agccgtgttt gtgccactgc 29400
actcctatct gggtgacaga acaagatcct atctcgaaaa taaatgcata ttcatatctt 29460
tatctgaaag tccatgtggc agtcagtcca tgtgtagatg tactacgatg gatatttatt 29520
gttcttattt ttaccctgat tgattgattt atttttttat tttatttttt ctgagacaga 29580
gtctcgctct gtcacccagg ctggagtgca gtggtgcgat ctcgggtcac tgcaagctcc 29640
gcctcccggg ttcacgccat tctcctgctt cagcctccca agtagctggg actacaggcg 29700
cccgccacaa cgcctggcta attttttttg tatttttagt agagacggtg tttcactgtg 29760
ttagccagga tggtctcgat ctcctaacct cgtgatctgc ccacctcagc ctcccaaagt 29820
gctggggtta caggcgtgag ccaccatgcc cagcctttat tatttttttt aagagacttt 29880
tgaatgtttt tatccctttg aagctcgctc tgtttgaatg tttttattcc tttgaagctt 29940
gctctgttgc ccaggctgga gtgcagtggt gcgatcatag ctcactgcaa cgtggatctt 30000
ctgatggata tttagattgc ttacattttc agtgctatga tcaatgtttt tatatagaca 30060
agtttttggg cttgtgctct ttgttacctt gtagtaagga cacgttccta ggataataat 30120
ggtgattttt gtgttttgga tatattatct catttaacac ttaggtcact ctatgggggt 30180
gactaccatc gtgtcaattg tgaagatcac aaaattgtac cataaaggac aattaagaaa 30240
tttgaccgga gttacacaat aaagaatgga caagctgaca tttatttttt ggagtatctt 30300
aattgggcct tcagtttgaa ggatgtttgt cctaggtcta aagtaggctg tgcttgttgt 30360
tcaatacttt gcaggaatca ttccattgct ttctggtctg tctggctgat agcctggaat 30420
tttttattga gtgctaggca ttgggtatga aaaaactgta gcgrtatttt gagggctgga 30480
tggtgttatt tttctctcca gagttgactt gtccttcttt tgggcagata aggtgtagga 30540
ggtatcttca ttctgtcagg aattgttgag cttcctaaag gctaaatagg aatttagcct 30600
tcagtctttg tgaggactgg tctgttcatg gttcattcct gctcctgaat ccctaggtat 30660
ctactgctst acgctgaact cggcttttgt cccttagtcc tctaagactg ccaaaagttg 30720
tgctcagcca atttaccttt agtttattgc atgcaacttg gaaaatgcct tgaggagaaa 30780
agcatagact gtcaggccca tttcttactg cttccatttt ctctgggatc tttgtctgtt 30840
agcacttgct gtctgtccgc tctgcccagc atttctgttt gttactaaaa gaggattggt 30900
ctcttaagct atttgctgta acagaaggag atgaggggct gcagcttgct cccaggtctg 30960
aatcctgagg ccttttcagt agaattgctg gaaataagcc ttaaaatctt tatccagaga 31020
gttctgtctg ttgataagca tacgacgatt aacatatctt ttttgtttga tttaatggat 31080
attctttcat ttctatcttt gacaattaat ggtgtcctga agtaaagaaa catgtaggca 31140
ttatacatag aaatatcttt cagccctttg gaatccactc tggttggttc atgtactttg 31200
aaaaatactg aatggatatt gacctagatt ccatggtacc atgttgagar ttgtgcctag 31260
ctactgagag tcttttttct aaggtttgta gatattggat tcattaataa tttagtgatc 31320
ttgataggtt ttgtgcctct ggatattttt tataagagtg gtaggtatga tcttaatcgt 31380
ttatctaaac tctttccttg cttcatatga attcttagta gaaatttgga aacttaagga 31440
tgagagaatg atagctttgt tctcctttct gttttacccg tctttttaag tggatttttg 31500
ttaacattcc agcctcagaa ttattaggtc tggaaccatt tcagtcctga gagagccaga 31560
caggtggatg gaatgtttat taatactgaa catgtttgaa ttatttcctc ttctcccatt 31620
tttcttgaat agtgctttaa ttgtgttaac tatttgtagc atgtttctaa gactggattc 31680
ccttactccc agtccactgg catttctcgt atgccttgat cccaacccag gatgccaagt 31740
gtattagttt ttatcacgct gctatgaaga aattcctgag actgggcaat ttataaagaa 31800
aagaggttta attgactcac agttccacac ggttggggag gcctcaggaa tcttataatc 31860
atggcagaag gcacctcttc agagggcggc aggagaggga atgagtgcaa gcgagagaaa 31920
tgccagacgc ttactgtcag atctcatgag actcattcat tgtcatgaga actacatggg 31980
ggaaactgcc cccatgatcc agttacctcc acctagtccc gcctgttgac acatggggat 32040
tattataatt cagggtgaga tttaggtggg gacacagcca aaccgtatca ccaagattat 32100
ctttgaaggt ctagacatct atgaagctgg cttttgctac taagttttct gggaaccaga 32160
ggaatcagat ttatagttga tgactcaaat agcagataaa gataaaagat atataacatt 32220
aataatacct tagagtaata aattcttaag aaatcctaat agcttttttt aaattaaact 32280
ttttaatttt gagataaaag ttgtagattc atatacagtt gtaagagttc ccatgtaacc 32340
gttaccctat ttctcctaat gctagcattt gtcacactac agtacagttc cacaaccatg 32400
ccattgacat tgataacagt taaagacaca gggcatttcc aacactgcag tgatccctca 32460
tgttcttttg tatccgggct cacttgatat ggcttccacc ctctccttac ccttgggcaa 32520
ccacaaatct gtttttcctt tctataattt tcattcatcg tgcattcatg tacagttttt 32580
gtgtgtgtgt gtgagcataa ggtttttgtc ttttggggaa acagggtcca actctgttgt 32640
ccaggctgga gtgcaatggc tcggtcttgg ctcactgcaa tctccgcctc ccgggttcaa 32700
gcgattctcc tgcctcagcc tcccaaggag ctgggattac aggcatgcgg caccacacct 32760
ggctaatctt tgtattttca gtagagacgg ggtttcacca tgttggtcag gctggtcttg 32820
aactcctgac tcaggtgatc tacccgcctg ggattacagg catgagccac tgtgcccagc 32880
ctaaacgtaa gtttttattt ctctttgata aatgcccagg gttgttaact gctttgtggt 32940
atggtagttg catgtttagt ttactaagaa actgctgaat tgttttccag agtggcttgt 33000
accattttgt attcccacca gcaatggatg agtgatccaa tgtctctgca tccttactag 33060
cttttggtat tactactttt tttttttttt taaagacatg ggatagttgt gtagcaatct 33120
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
ctcactgatt ttaatttgca taatagctca tgatgttgaa catttttttg tgtacttatt 33180
tgctacatgt gtatcctttt tggtgaaaat gtctgtcttt tgcccatttt ctaattaaat 33240
ttttgattgt tactgagttt ttttgttttg ttttgagatg aagtctcgct ctgtcaccta 33300
gactggagtg cagtggcatg atcttgtctc actgcagcct ccaccacccg ggttcaagtg 33360
gttctcctgt ctcagcttcc caagtagctg ggattacagg aatgcgccac cacacctggc 33420
aaatttttat atttttagta gagatggggt tcaccatgtt ggtcaggctg gcctggaact 33480
tctgacctca ggtggtccac ccgccttggc ctcccaaagt gctaggatta caggcatgtg 33540
ccactgcgcc tgtcctgtta cagagttttt aaagtttttt tatgttttag gtgctagttt 33600
gttgttggat atgaggtttg ttaataattt tctcctagtt cgtagcttgt cttttcatcc 33660
tctttaatga ggaatctttt acagaggaaa agttcttaat tttactgaag tcaaatttat 33720
caacttatct gttggctgta gcaaatatcc tctgtccatt tagcatacag acgtggacta 33780
tcatccttct tttccatttg tattcatgcc tgatactaaa gatagtgata tttttgggcg 33840
ttattcttgt tgttctattt ggactggtaa ttcctaatgg tttttgaaca gtttcaactt 33900
aagttattct aaacaaaaga gaaaatgatc gtaatttgat ttttccaaag tcttggctgt 33960
ctgtgtgcat cataacgatt tgtgatgtct atgaaacaat tctccgcatt ggttctgaac 34020
gtaaatttta ccatgtctcc agatatttaa aagtactctg gtcaagaaaa tattgtttgt 34080
cagttttgga atggatatgg tagttcttac ctcatgtgat ataaaaaggc ttaatccaca 34140
attgctacaa aataacatga gtttgtgtct atggatgcat tgtgctaagt tccaccccaa 34200
atgtgttaaa tatttaagta aatcatggta gtgtgtagga gaacagttaa tttccctctg 34260
tcatttgagt ggtataacta tgttgtaggt cttttacatt aaatagaact aagaaactta 34320
aagttggcca ggtgtggtgg ctcacacctg ttgtaatcct ggcactctgg gaggctgagg 34380
cacgtagatg acttgagctc aggagttcga gaccagcctg gccaacatgg tgaaacccca 34440
tctctactaa aaatacaaaa attagccagg catggtggtg catgcgcctg taattgaagc 34500
gtgagaatct cttgcacccg ggaggtggag gttacagtga gccgagattg tgccgttgga 34560
gattgtccca ttgtgcctgg gagacagagt gagactctga ctcaaaaaaa ccacaaaaaa 34620
taatatttaa aaaaagaagc ttaaagttaa atgagtttaa aaggccccat gcaatttacc 34680
attgtgtatg tgtttcagtt gccagagtct gccctccgag acctcatcgt agccaccatt 34740
gctgtcaagt acactcagtc taactctgtg tgctacgcca agaacgggca ggtaagtggg 34800
ctgttggact cgccttcggg ggactgttgt tttacgaaat gatatttaaa catccgtctg 34860
ccttagatat tggacagctt gaaagggaat atttgccaaa tgtttgtctt ttgtgtttgt 34920
cagtgtttcc tcagtaccct ggtgggctgt taaaactaat gatgatcaga aaatatcttt 34980
ggaaccaggt actcaaagta gttgtgctgc ttcttgcctt gaataggttg gcacattttg 35040
gattagtgag actctaatag ctgttacaaa actggattat ttcctttcct tttcccccat 35100
tggtattatt tccagggaag aggaggtaag ttacggtact gccacccrcc agcctatatt 35160
tttgcagcct gcagggtatt ttcacattat ttcttatgtt gtcttatcaa gaatctgctg 35220
agtagaaaga ctgctagact aggagttaac ttcatcttta aaaagttagg ttcattgagg 35280
cataatttgt atacagtaga attcaccttt ttaaagtaca gtttgaccag ttttcacaaa 35340
tttcacagta tgtgaccgct gccacaatta aataccaaac atttctcact gtgtgacgtt 35400
aagcaagata ttacacttct ttcctagtgt cccgagcctc agaatacaag atggagctgg 35460
ttcatctctg aggtctctgc tagccttttc ttcacatggt gtcctgagct tatctcttgg 35520
catttgttaa attatttggc ccacaagtga cagggaggtg ggtggatatg aattagccta 35580
tggcctggaa gaccccagat tgccagtctc tgtagtaaca aactcctggg agattgtgtc 35640
tactggtgtc aggttctaag aagttctgta agtccatgta gaatgagtgc ttcattcctg 35700
atgttgagga ggaggcggct gcccaggtat ttgtggtctc ttttattctc ttgtagctgt 35760
gggcagggtt gccagcagct gtgctcagaa agagatggac tgttgggcag acagcttagg 35820
ggctgttcat tataggaact aaaaccagaa aacacacctg tgcatttaag tccttgttgc 35880
ttagaattca acccatagaa atagaggaaa gaaggaagaa catttgtacc tggcggaatt 35940
gtttctactt ttagctaccc atgtcagatc cttatctcaa tttctgagag ttgatatttt 36000
aactgtaatt tatacactgg ggcaaacaaa agactttcaa gtaaccatag ttatttttgt 36060
ggtcattagt tttatttatt tatttattta ttttttatag agacagggtc ttgctctgtt 36120
gcccaggcca gagtacagtg acatgatcat agctcattgt agccttcgac tcctggcctc 36180
aagcggtcct accacttcag cctcctgagt agctgggact gcaggtgtgc gccactaccc 36240
ctagctattt tttaattctt attttttgta gagatgagat gttgccatct tgcccaggct 36300
ggtctcctga gctcaagcaa tccttctgtc ctaacctcac aaagtgctgg gattacaggc 36360
atgagccagt gcacccagca ttggtcattt attttaactt tttttttttt ttgacctcaa 36420
accatcaatt ttattgaatt gtagattatt aatagtcata tgtcacatat cactgaagag 36480
gtataaatag tagttataaa caaacctctt actccacccc ttttaatgac ttaaaaatta 36540
tttatatttc tcttattttg gcctgatatg ttcttccaca aaactaataa ataggttttg 36600
gagaatgtgc acaaaagatc cttttgagaa gaaagtgtct ttggaagagg tgttcacttt 36660
aatgtctgtg ttcctcaggt tatcggcatt ggagcaggac agcagtctcg tatacactgc 36720
actcgccttg caggagataa ggcaaactat tggtggctta gacaccatcc acaagtgctt 36780
tcgatgaagt ttaaaacagg rgtgaagaga gcagaaatct ccaatgccat cgatcaatat 36840
gtgactggaa ccattggcga ggtgaaagac ttggcattgg gttctcggct gtgttaatat 36900
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
21
tcagttcatc cctttatgtg taacagattt taacttcatc accacacaga gaaaaagata 36960
ctttcattga aattctatgt tgtctaaaat tgatcattga aacttcttac acatttctca 37020
tgttcttctg tctcatgtaa ctttcttcat tgtttttgac ccctttctga aaccacagtc 37080
ctctgtcttt ttaaattgca gccttggtgt aggtttgtat atttgtactt cccctgttga 37140
ttataagctt ttgatgacaa ggactgcttt tcacctacgt cgaggtgcct gccaccacgt 37200
catgcatggt gcttgctgtc cgatgggtct gtattcaaca ttaaatacaa attgcctgac 37260
ggcaggtaac cctggggtct tctccyacca cattttctac atgtgccatt aataacttga 37320
tacattttgt tacatttcat gttcttctgt ttgaagaagg tatgtgggaa cattacaatc 37380
gcaaacgtta ggtaggtagc ccgccagaag aaaagatcta gcccagtttc tggacatatt 37440
gcgtgcctaa caaaatgctg gatggaaaac agaagcctgc taatcaaata ctagatggtc 37500
ttctgaagag ccaattgact accctcagtt ttttaytcag gagcaggaat caacataagt 37560
gtcattgaag agtgaggctg cagaatgtaa ctgaagtcct taagatatct tttttttttc 37620
aactttgata cttggatttt tctgtttgat attaccagtc aacccctgct actagtcctg 37680
aaagagtcct ggaagtgaac aagaataaaa gtatgtcaaa gctaaaacat gacccaagat 37740
agaagcacct tgtaaaatat gattaactgt ctgtgccact atgacacaat actgcaggct 37800
gggtaactta taaaccatag acatttattt ttcatagttc tggaggctgt gaagtcccag 37860
atcaaggtac tggtgggctg agagtctggg gaggcctacc ctctgcttcc cagatggcac 37920
agccctctct ggaaaggaca aacactgtgt ccttacttca cagaaggggc,atacttcccc 37980
tgaagccctt ttataagtct gtaatccatt catgagggcc ccaccctcat tgcccaataa 38040
ctttctaaag gccttatact tcttaatact gttgcattgg ggattaagtt tcaacatgaa 38100
ttttggagtg gatacacaca ctcaaaccat agcaaatatt atgaattgtg ttttgtctac 38160
aactgccttt acatttagaa taatttttgt ataaatgtag ttaaagaact actcatctct 38220
ttcctagatt ggatatagag ggtttttttg aaaacccttg gaacacaatt tttatttgct 38280
ttctttgcta cagtagtacc aaagcagtag atttaaatta gacggtagtc taatagacat 38340
aaagtagtag atacgaagta gatttaaatt agagatttta aaacagtgta tttttatgta 38400
agtagcttag ccaaaaataa aaaaaattac atatgtactt ggaataaact gatttggaaa 38460
gtaaaatggt attatcttta tttggtaaac ttagaatcac tttttttcct taatgaaaat 38520
atttctaaaa gcatcaaaga gattctagat atgtgaactt ccatgtaaat aatggtcatt 38580
atttacaatt aagaaatcct ggccgggcgc ggtggctcat gcctataatc ccagcacttt 38640
gggaggccga ggtgagtgga tcatgaggtc aagagattga gaccctcctg gccaacatgg 38700
tgaaactctg tctccaaaaa tacaaaaatt agctgggtgt ggtggtgtgc acttgtggtc 38760
ccaggtactt gggaggctga ggcaggagaa ttgcttgaac ctgggaggtg gaggttgcgg 38820
tgagccgaga tcgcaccact gcactccagc ctggtgatag tgcaaaactc cgtctaaaaa 38880
aaaaataata ataataataa taaaaacaag tcctaagaaa aatgcccagg tgctttctgg 38940
catggtgatt tgcaccacat agaactaaag acgatgtcag accaagcttc ttcctttctc 39000
tctccccgca taggatgaag atttgataaa gtggaaggca ctgtttgagg aagtccctga 39060
gttactcact gaggcagaga agaaggaatg ggttgagaaa ctgactgaag tttctatcag 39120
ctctgatgcc ttcttccctt tccgagataa cgtagacaga gctaaaaggg taagtatgga 39180
attgggtgca tttgcttaga gttgagcatt atgtagaaac tgtttcagaa atcctgcttt 39240
tgatttttaa aaggtgtggc aaagtgatac agatcagtaa tattcagaga accatttgac 39300
ttctccattg ggtggatgga raacccaaat cctgttgtta ttttgccttt ttgactgagt 39360
gtatctttgt tagcatatgc tttttagagg gggattttga gttttgcagg tttttacata 39420
aaatcgcgtt ttgaaaatca atatacttcc cccagagtgg tgtggcgtac attgcggctc 39480
cctccggttc tgctgctgac aaagttgtga ttgaggcctg cgacgaactg ggaatcatcc 39540
tcgctcatac gaaccttcgg ctcttccacc actgatttta ccacacactg ttttttggct 39600
tgcttatgtg taggtgaaca gtcacgcctg aaactttgag gataactttt taaaaaaata 39660
aaacagtatc tcttaatcac tggatccaka gtttttggta gttgtgtttt atgttaaaga 39720
tgcaggctct ttgaactgac acatgacaca taacacataa atgaggaatt ccagagcacc 39780
cctgcctacc ggagctcagc ccatcccaca gcactgcccg tgtgaaacat aaacattagc 39840
aggaaccaaa cgagctgagc agccagagga catggcacaa gtcactgtgt acaggccaca 39900
cttaaggact gggagttata cccatcttaa aggtggagta ttgatgtaaa ttacctagaa 39960
ttcttctgca tgggagttgt atattaaggg tccatgttgc ctcctagggg agtcttctca 40020
tgccgttttt gtttgtttgt ttttcatttt tttgatttgt tattttgaga gggagtctca 40080
aaaaaatagc tctgtcactc aggctagagt cagtggcacc gtcttggctc actgcaacct 40140
ccgcctcccg ggttcaagcg attctcctgc ctcagtctcc tgagtagctg ggactacagg 40200
catgtgccac catgcctggc taatttttgt atttagtaga gacagggttt caccatgttg 40260
gtcaggctgg tctcgaactc ttgacctcat gatccacctg ccctggcctc ccaaagtgct 40320
gggattacgg gcgtgagcca ccgtgcccgg ccctcatgcc atttattttt aatcacactt 40380
ctgagaagct tggttgtcta ctttccaaac aaacagcaga ttggcacctg tgaactggaa 40440
ccttagaggg gattggttta agtcttgttg accccctcta tggataatct gatgtatatt 40500
tttctcagtg ctaagtgaaa tgtttcccag aatttcagca gcccgagatt caccctctgg 40560
agctgcataa aaatgtagtc aatatttggt gctcagaaat tgtacccaat attccaatta 40620
caggcttaat cactacagtg ggcacagtgg gagggcagtg ccttccttca tcaggacaga 40680
CA 02368672 2001-09-24
WO 00/56924 PCT/IBOO/00404
22
cctgtgcatc tgtgtgtcct gcctgtgtgc tcctgaccat tcccgtgcat tgcacctgtg 40740
ttcaactaaa accctttgcc attgttcacc tttaccaatg agtcctaccc tcttcccaag 40800
tctttaactt atccccttgc taaataaaaa tttctgaagt tttttcttaa tggtcatctt 40860
tctaagttat ttctggttca gctaatcttc cagccctggc ctaggacctg cccacattga 40920
aacaaggctg acttgtgtga tcagtaaaat actgcagaaa aaataagact tttgaggcta 40980
ggtcctaata cattgcaact tcagccttag tttcttggat tacttcctct gggataagcc 41040
aagaccacgt tgtaagagtt aagcagccct ccacaaggag agacgccatc ttgcttacac 41100
tagttgccag ccacatgagt gagccacctc agaggtggat cctccagctt cagtcccagg 41160
caacatctgg cttcaacctc ttcagagacc tgagccagaa ctgcccaaaa gagctcttga 41220
attcctgacc cacagataca gagagatgca tactgttgtt aagccacaaa gttctggggt 41280
aattatgtag cagtaaatag ctaatacaga ttttggcttg taaattaagt gtgtgttgtc 41340
'tttttcatgg ttctttggct tgaccaaagg ttaacattaa gggtatgata atgggaacag 41400
gctgagcact gtgtctcctg tctataatcc cagcactttg ggaggattgc ttgaggccag 41460
gagttcaaga ccagcctggg caacatagcg acatcctcat ctctaaaaaa agagaaaatt 41520
ttaattagct gggcgtggtg gctcctgttt gtagtgttct tcacatagat gaagaataaa 41580
taagtggaga atatgcaact cccatgcaga agaattctag gtaatttaca tcaatactct 41640
actgaggggt ataactccca gtccttaagt gtggcctgta caca 41684
<210> 2
<211> 1965
<212> DNA
<213> Homo sapiens
<220>
<221> 5'UTR
<222> 1..77
<220>
<221> CDS
<222> 78..1856
<220>
<221> 3'UTR
<222> 1857..1965
<220>
<221> polyAsignal
<222> 1938..1943
<220>
<221> allele
<222> 424
<223> 99-5602-372 : polymorphic base G or C
<220>
<221> allele
<222> 1520
<223> 5-297-209 : polymorphic base A or C
<400> 2
cccggcagcc ctcctacctg cgcacgtggt gccgccgctg ctgcctcccg ctcgccctga 60
acccagtgcc tgcagcc atg gct ccc ggc cag ctc gcc tta ttt agt gtc 110
Met Ala Pro Gly Gln Leu Ala Leu Phe Ser Val
1 5 10
tct gac aaa acc ggc ctt gtg gaa ttt gca aga aac ctg acc gct ctt 158
Ser Asp Lys Thr Gly Leu Val Glu Phe Ala Arg Asn Leu Thr Ala Leu
15 20 25
ggt ttg aat ctg gtc gct tcc gga ggg act gca aaa gct ctc agg gat 206
Gly Leu Asn Leu Val Ala Ser Gly Gly Thr Ala Lys Ala Leu Arg Asp
30 35 40
gct ggt ctg gca gtc aga gat gtc tct gag ttg acg gga ttt cct gaa 254
Ala Gly Leu Ala Val Arg Asp Val Ser Glu Leu Thr Gly Phe Pro Glu
45 50 55
CA 02368672 2001-09-24
WO 00/56924 PCT/IBOO/00404
23
atg ttg ggg gga cgt gtg aaa act ttg cat cct gca gtc cat gct gga 302
Met Leu Gly Gly Arg Val Lys Thr Leu His Pro Ala Val His Ala Gly
60 65 70 75
atc cta gct cgt aat att cca gaa gat aat gct gac atg gcc aga ctt 350
Ile Leu Ala Arg Asn Ile Pro Glu Asp Asn Ala Asp Met Ala Arg Leu
80 85 90
gat ttc aat ctt ata aga gtt gtt gcc tgc aat ctc tat ccc ttt gta 398
Asp Phe Asn Leu Ile Arg Val Val Ala Cys Asn Leu Tyr Pro Phe Val
95 100 105
aag aca gtg gct tct cca ggt gta ast gtt gag gag gct gtg gag caa 446
Lys Thr Val Ala Ser Pro Gly Val Xaa Val Glu Glu Ala Val Glu Gln
110 115 120
att gac att ggt gga gta acc tta ctg aga gct gca gcc aaa aac cac 494
Ile Asp Ile Gly Gly Val Thr Leu Leu Arg Ala Ala Ala Lys Asn His
125 130 135
gct cga gtg aca gtg gtg tgt gaa cca gag gac tat gtg gtg gtg tcc 542
Ala Arg Val Thr Val Val Cys Glu Pro Glu Asp Tyr Val Val Val Ser
140 145 150 155
acg gag atg cag agc tcc gag agt aag gac acc tcc ttg gag act aga 590
Thr Glu Met Gln Ser Ser Glu Ser Lys Asp Thr Ser Leu Glu Thr Arg
160 165 170
cgc cag tta gcc ttg aag gca ttc act cat acg gca caa tat gat gaa 638
Arg Gln Leu Ala Leu Lys Ala Phe Thr His Thr Ala Gln Tyr Asp Glu
175 180 185
gca att tca gat tat ttc agg aaa cag tac agc aaa ggc gta tct cag 686
Ala Ile Ser Asp Tyr Phe Arg Lys Gin Tyr Ser Lys Gly Val Ser Gln
190 195 200
atg ccc ttg aga tat gga atg aac cca cat cag acc cct gcc cag ctg 734
Met Pro Leu Arg Tyr Gly Met Asn Pro His Gln Thr Pro Ala Gln Leu
205 210 215
tac aca ctg cag ccc aag ctt ccc atc aca gtt cta aat gga gcc cct 782
Tyr Thr Leu Gln Pro Lys Leu Pro Ile Thr Val Leu Asn Gly Ala Pro
220 225 230 235
gga ttt ata aac ttg tgc gat gct ttg aac gcc tgg cag ctg gtg aag 830
Gly Phe Ile Asn Leu Cys Asp Ala Leu Asn Ala Trp Gln Leu Val Lys
240 245 250
gaa ctc aag gag gct tta ggt att cca gcc gct gcc tct ttc aaa cat 878
Glu Leu Lys Glu Ala Leu Gly Ile Pro Ala Ala Ala Ser Phe Lys His
255 260 265
gtc agc cca gca ggt gct gct gtt gga att cca ctc agt gaa gat gag 926
Val Ser Pro Ala Gly Ala Ala Val Gly Ile Pro Leu Ser Glu Asp Glu
270 275 280
gcc aaa gtc tgc atg gtt tat gat ctc tat aaa acc ctc aca ccc atc 974
Ala Lys Val Cys Met Val Tyr Asp Leu Tyr Lys Thr Leu Thr Pro Ile
285 290 295
tca gcg gca tat gca aga gca aga ggg gct gat agg atg tct tca ttt 1022
Ser Ala Ala Tyr Ala Arg Ala Arg Gly Ala Asp Arg Met Ser Ser Phe
300 305 310 315
ggt gat ttt gtt gca ttg tcc gat gtt tgt gat gta cca act gca aaa 1070
Gly Asp Phe Val Ala Leu Ser Asp Val Cys Asp Val Pro Thr Ala Lys
320 325 330
att att tcc aga gaa gta tct gat ggt ata att gcc cca gga tat gaa 1118
Ile Ile Ser Arg Glu Val Ser Asp Gly Ile Ile Ala Pro Gly Tyr Glu
335 340 345
gaa gaa gcc ttg aca ata ctt tcc aaa aag aaa aat gga aac tat tgt 1166
Glu Glu Ala Leu Thr Ile Leu Ser Lys Lys Lys Asn Gly Asn Tyr Cys
350 355 360
gtc ctt cag atg gac caa tct tac aaa cca gat gaa aat gaa gtt cga 1214
Val Leu Gln Met Asp Gln Ser Tyr Lys Pro Asp Glu Asn Glu Val Arg
365 370 375
act ctc ttt ggt ctt cat tta agc cag aag aga aat aat ggt gtc gtc 1262
Thr Leu Phe Gly Leu His Leu Ser Gln Lys Arg Asn Asn Gly Val Val
380 385 390 395
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
24
gac aag tca tta ttt agc aat gtt gtt acc aaa aat aaa gat ttg cca 1310
Asp Lys Ser Leu Phe Ser Asn Val Val Thr Lys Asn Lys Asp Leu Pro
400 405 410
gag tct gcc ctc cga gac ctc atc gta gcc acc att gct gtc aag tac 1358
Glu Ser Ala Leu Arg Asp Leu Ile Val Ala Thr Ile Ala Val Lys Tyr
415 420 425
act cag tct aac tct gtg tgc tac gcc aag aac ggg cag gtt atc ggc 1406
Thr Gln Ser Asn Ser Val Cys Tyr Ala Lys Asn Gly Gin Val Ile Gly
430 435 440
att gga gca gga cag cag tct cgt ata cac tgc act cgc ctt gca gga 1454
Ile Gly Ala Gly Gln Gln Ser Arg Ile His Cys Thr Arg Leu Ala Gly
445 450 455
gat aag gca aac tat tgg tgg ctt aga cac cat cca caa gtg ctt tcg 1502
Asp Lys Ala Asn Tyr Trp Trp Leu Arg His His Pro Gln Val Leu Ser
460 465 470 475
atg aag ttt aaa aca ggr gtg aag aga gca gaa atc tcc aat gcc atc 1550
Met Lys Phe Lys Thr Gly Val Lys Arg Ala Glu Ile Ser Asn Ala Ile
480 485 490
gat caa tat gtg act gga acc att ggc gag gat gaa gat ttg ata aag 1598
Asp Gln Tyr Val Thr Gly Thr Ile Gly Glu Asp Glu Asp Leu Ile Lys
495 500 505
tgg aag gca ctg ttt gag gaa gtc cct gag tta ctc act gag gca gag 1646
Trp Lys Ala Leu Phe Glu Glu Val Pro Glu Leu Leu Thr Glu Ala Glu
510 515 520
aag aag gaa tgg gtt gag aaa ctg act gaa gtt tct atc agc tct gat 1694
Lys Lys Glu Trp Val Glu Lys Leu Thr Glu Val Ser Ile Ser Ser Asp
525 530 535
gcc ttc ttc cct ttc cga gat aac gta gac aga gct aaa agg agt ggt 1742
Ala Phe Phe Pro Phe Arg Asp Asn Val Asp Arg Ala Lys Arg Ser Gly
540 545 550 555
gtg gcg tac att gcg gct ccc tcc ggt tct gct gct gac aaa gtt gtg 1790
Val Ala Tyr Ile Ala Ala Pro Ser Gly Ser Ala Ala Asp Lys Val Val
560 565 570
att gag gcc tgc gac gaa ctg gga atc atc ctc gct cat acg aac ctt 1838
Ile Glu Ala Cys Asp Glu Leu Gly Ile Ile Leu Ala His Thr Asn Leu
575 580 585
cgg ctc ttc cac cac tga ttttaccaca cactgttttt tggcttgctt 1886
Arg Leu Phe His His *
590
atgtgtaggt gaacagtcac gcctgaaact ttgaggataa ctttttaaaa aaataaaaca 1946
gtatctctta atcactgga 1965
<210> 3
<211> 592
<212> PRT
<213> Homo sapiens
<220>
<221> VARIANT
<222> 116
<223> Xaa=Thr or Ser
<400> 3
Met Ala Pro Gly Gin Leu Ala Leu Phe Ser Val Ser Asp Lys Thr Gly
1 5 10 15
Leu Val Glu Phe Ala Arg Asn Leu Thr Ala Leu Gly Leu Asn Leu Val
20 25 30
Ala Ser Gly Gly Thr Ala Lys Ala Leu Arg Asp Ala Gly Leu Ala Val
35 40 45
Arg Asp Val Ser Glu Leu Thr Gly Phe Pro Glu Met Leu Gly Gly Arg
50 55 60
Val Lys Thr Leu His Pro Ala Val His Ala Gly Ile Leu Ala Arg Asn
65 70 75 80
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
Ile Pro Glu Asp Asn Ala Asp Met Ala Arg Leu Asp Phe Asn Leu Ile
85 90 95
Arg Val Val Ala Cys Asn Leu Tyr Pro Phe Val Lys Thr Val Ala Ser
100 105 110
Pro Gly Val Xaa Val Glu Glu Ala Val Glu Gln Ile Asp Ile Gly Gly
115 120 125
Val Thr Leu Leu Arg Ala Ala Ala Lys Asn His Ala Arg Val Thr Val
130 135 140
Val Cys Glu Pro Glu Asp Tyr Val Val Val Ser Thr Glu Met Gin Ser
145 150 155 160
Ser Glu Ser Lys Asp Thr Ser Leu Glu Thr Arg Arg Gln Leu Ala Leu
165 170 175
Lys Ala Phe Thr His Thr Ala Gln Tyr Asp Glu Ala Ile Ser Asp Tyr
180 185 190
Phe Arg Lys Gln Tyr Ser Lys Gly Val Ser Gln Met Pro Leu Arg Tyr
195 200 205
Gly Met Asn Pro His Gln Thr Pro Ala Gln Leu Tyr Thr Leu Gln Pro
210 215 220
Lys Leu Pro Ile Thr Val Leu Asn Gly Ala Pro Gly Phe Ile Asn Leu
225 230 235 240
Cys Asp Ala Leu Asn Ala Trp Gln Leu Val Lys Glu Leu Lys Glu Ala
245 250 255
Leu Gly Ile Pro Ala Ala Ala Ser Phe Lys His Val Ser Pro Ala Gly
260 265 270
Ala Ala Val Gly Ile Pro Leu Ser Glu Asp Glu Ala Lys Val Cys Met
275 280 285
Val Tyr Asp Leu Tyr Lys Thr Leu Thr Pro Ile Ser Ala Ala Tyr Ala
290 295 300
Arg Ala Arg Gly Ala Asp Arg Met Ser Ser Phe Gly Asp Phe Val Ala
305 310 315 320
Leu Ser Asp Val Cys Asp Val Pro Thr Ala Lys Ile Ile Ser Arg Glu
325 330 335
Val Ser Asp Gly Ile Ile Ala Pro Gly Tyr Glu Glu Glu Ala Leu Thr
340 345 350
Ile Leu Ser Lys Lys Lys Asn Gly Asn Tyr Cys Val Leu Gin Met Asp
355 360 365
Gln Ser Tyr Lys Pro Asp Glu Asn Glu Val Arg Thr Leu Phe Gly Leu
370 375 380
His Leu Ser Gln Lys Arg Asn Asn Gly Val Val Asp Lys Ser Leu Phe
385 390 395 400
Ser Asn Val Val Thr Lys Asn Lys Asp Leu Pro Glu Ser Ala Leu Arg
405 410 415
Asp Leu Ile Val Ala Thr Ile Ala Val Lys Tyr Thr Gln Ser Asn Ser
420 425 430
Val Cys Tyr Ala Lys Asn Gly Gln Val Ile Gly Ile Gly Ala Gly Gln
435 440 445
Gln Ser Arg Ile His Cys Thr Arg Leu Ala Gly Asp Lys Ala Asn Tyr
450 455 460
Trp Trp Leu Arg His His Pro Gln Val Leu Ser Met Lys Phe Lys Thr
465 470 475 480
Gly Val Lys Arg Ala Glu Ile Ser Asn Ala Ile Asp Gln Tyr Val Thr
485 490 495
Gly Thr Ile Gly Glu Asp Glu Asp Leu Ile Lys Trp Lys Ala Leu Phe
500 505 510
Glu Glu Val Pro Glu Leu Leu Thr Glu Ala Glu Lys Lys Glu Trp Val
515 520 525
Glu Lys Leu Thr Glu Val Ser Ile Ser Ser Asp Ala Phe Phe Pro Phe
530 535 540
Arg Asp Asn Val Asp Arg Ala Lys Arg Ser Gly Val Ala Tyr Ile Ala
545 550 555 560
Ala Pro Ser Gly Ser Ala Ala Asp Lys Val Val Ile Glu Ala Cys Asp
565 570 575
Glu Leu Gly Ile Ile Leu Ala His Thr Asn Leu Arg Leu Phe His His
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
26
580 585 590
<210> 4
<211> 450
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 78
<223> 99-22578-78 : polymorphic base C or T
<220>
<221> miscbinding
<222> 66..90
<223> 99-22578-78.probe
<220>
<221> primerbind
<222> 59..77
<223> 99-22578-78.mis
<220>
<221> primer_bind
<222> 79..97
<223> 99-22578-78.mis complement
<220>
<221> primer_bind
<222> 1..18
<223> 99-22578.pu
<220>
<221> primer_bind
<222> 430..450
<223> 99-22578.rp complement
<400> 4
tgccccttga aaatctacac tccaaatgag tacattacaa ctatggtgca atgagtgatt 60
ttccccaagg taccatgytc attggtttcc acaggacagg caacctagca gggcattccc 120
tccatgaggt tatgaaaaca cgctgtgctc ctgtagaccc acacacagca ccctccccat 180
tgtacttatt gccaaacact gggcttccta atcactttgt gttcagtcag agatccagga 240
aatccaaacc cagccagaaa aatgcacaac agctcagcat aagcagcttt aataggagct 300
taaggaagct tccattgctc ccatcctgga aaagcatgtg ttgtagcaga aagagcacaa 360
gctctagcaa tggacaggcc tcaggtcata tcctggatct ggctgggttg ctgggttacg 420
tgctgggttg tctcagataa ggtcaagtct 450
<210> 5
<211> 506
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 72
<223> 99-22580-72 : polymorphic base A or T
<220>
<221> misc_binding
<222> 60..84
<223> 99-22580-72.probe
<220>
CA 02368672 2001-09-24
WO 00/56924 PCT/IBOO/00404
27
<221> primerbind
<222> 53..71
<223> 99-22580-72.mis
<220>
<221> primerbind
<222> 73..91
<223> 99-22580-72.mis complement
<220>
<221> primer_bind
<222> 1..18
<223> 99-22580.pu
<220>
<221> primer_bind
<222> 488..506
<223> 99-22580.rp complement
<400> 5
tctcaggaaa tggacaacat attgtcaagt ttgaaagcat atggagctaa acgggattct 60
agtaaaggct cwtggtctta taatccaata tttataagca attaaagttc accaaagtct 120
ataaaaacat actgcagctg tgaatcaaat tagtgccttg acctacccaa ttagacaaag 180
aaaacatcaa tataataatt aggcagacaa tttccatctt agaattaatg taaatagtga 240
ttatgcctta aaaacaaatg ccgtattttt caaactagga gaaaattcat gtgctaaaag 300
atacaacatc ccaggttaga gagagtacct ccatgtttga ttagtgaatt gacaaggaga 360
attgtttttt ggtcactcag caaaattttc cttttgattt caattagtct ctctctcctt 420
ttacaaggat tgactgtcca tagattgaaa gtcattgctt tgtcagttca ggtttaaaga 480
gcaaagagtt tcaagccttc taatag 506
<210> 6
<211> 514
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 462
<223> 99-22585-462 : polymorphic base G or C
<220>
<221> miscbinding
<222> 450._474
<223> 99-22585-462.probe
<220>
<221> primer_bind
<222> 443..461
<223> 99-22585-462.mis
<220>
<221> primer_bind
<222> 463..481
<223> 99-22585-462.mis complement
<220>
<221> primer_bind
<222> 1..21
<223> 99-22585.pu
<220>
<221> primer_bind
<222> 494..514
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
28
<223> 99-22585.rp complement
<400> 6
gtagttactt ccattatctt cataatgaga atattgaggg gtgtacacaa cttgtctaaa 60
tgcacataac tattaagtga ataagtcagg gaacaaactc aggaagtctg acactataac 120
tcttaatgat caaggtacat tttcgtccat gtaatgatga taatactcat ctacctcaag 180
ccttgtggca aaatatagca aaagtagctt ggaaaatgta aagagctata gtaaaatgtc 240
ttatcaattt aactgcaaaa gaattttgaa aagacacgtg gtttgaataa tttacctctg 300
gattatcttt ggtttatgat ccaaggaaaa gaggacctca tggaaaaatc tttcagggtg 360
cttagctact ctttccagaa actgcttctg tccatctggg cacatgcacg gccagttctt 420
caagagtaga tgttgcctgg gacttgccac tggaattttt csttaaaatg ttaaaacagt 480
atttaattca catggtttgg tggaaaaatg aagt 514
<210> 7
<211> 497
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 347
<223> 99-23437-347 polymorphic base A or G
<220>
<221> misc_binding
<222> 335..359
<223> 99-23437-347.probe
<220>
<221> primer_bind
<222> 328..346
<223> 99-23437-347.mis
<220>
<221> primer_bind
<222> 348..366
<223> 99-23437-347.mis complement
<220>
<221> primer_bind
<222> 1..20
<223> 99-23437.pu
<220>
<221> primer_bind
<222> 478..497
<223> 99-23437.rp complement
<400> 7
cccatttcaa tcttagatag ctcttaccgt taggaagttc atctttatag tgaaataaaa 60
tcctacctcc ctgtaatttt tatctttagt catgaaagtc aaactgtact taagatgtgg 120
ttattttgtt cttaccttgc tagttatagt ttaattacca gtctttaagc actgtgaaaa 180
ttctaacatt ctcattctat caaactacat tctacattgt acagcaattt gtatctccat 240
agaaacaatt ccaacacata gaattgtaat tcccaaatgg cataattgta aacattttct 300
cagataactt caaagccatt tctgaaattt cttctaaaac attcacrtga actcagattg 360
tgaaaatgag ttatacctcc tttgaaatca agtcgttttt taattcctcc aaatataaat 420
gttaaaaact aaaatgtcaa aataagcaat ggtagtatta acacagttaa tactgaaggt 480
aaatgttaaa cacatgc 497
<210> 8
<211> 448
<212> DNA
<213> Homo sapiens
CA 02368672 2001-09-24
WO 00/56924 PCT/IBOO/00404
29
<220>
<221> allele
<222> 273
<223> 99-23440-274 polymorphic base A or G
<220>
<221> misc binding
<222> 261._285
<223> 99-23440-274.probe
<220>
<221> primer_bind
<222> 254..272
<223> 99-23440-274.mis
<220>
<221> primer_bind
<222> 274..292
<223> 99-23440-274.mis complement
<220>
<221> primer_bind
<222> 1..21
<223> 99-23440.pu
<220>
<221> primer_bind
<222> 428..448
<223> 99-23440.rp complement
<400> 8
gtggcttttt tccagtaaag gttaattatt aagaccacta gtcctggcct gggtcaatcc 60
cagtatgatc ctgggcaagt aaattaaaga agataacttc tctgtgcctc agtttttttt 120
tgttttgttt tttgtttttt catttacaaa atggagataa ttgtagtaaa tcaaattttt 180
agaggtgata ggtttgttca tttcttgaat gcggtgatgg tctcccaagt cacacatatg 240
taaaaaccca tcactttaaa gatatgcagt acrttgtatg acaagaaatt gcttttaaaa 300
ggagcaaact accttccagg gttgttgtga ggcataaatg gcaatccaca gcaccacagc 360
aaggattatc atgtgccctc cagagacata ctctcaggtg gatgcgagaa atatccagct 420
gttgcagcaa cttcatccca ctcgaaat 448
<210> 9
<211> 457
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 190
<223> 99-23442-190 polymorphic base C or T
<220>
<221> misc_binding
<222> 178..202
<223> 99-23442-190.probe
<220>
<221> primer_bind
<222> 171..189
<223> 99-23442-190.mis
<220>
<221> primer bind
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
<222> 191..209
<223> 99-23442-190.mis complement
<220>
<221> primer_bind
<222> 1..20
<223> 99-23442.pu
<220>
<221> primer_bind
<222> 437..457
<223> 99-23442.rp complement
<220>
<221> allele
<222> 396
<223> 99-23442-396 : polymorphic base A or C
<220>
<221> miscbinding
<222> 384._408
<223> 99-23442-396.probe
<220>
<221> primer_bind
<222> 377..395
<223> 99-23442-396.mis
<220>
<221> primer_bind
<222> 397..415
<223> 99-23442-396.mis complement
<400> 9
cttttgagta tagaaacccc tagtaacaat ttaagttcct tccatttttc ttttaaactc 60
cttattccca gcagcagtat tctacattct aaccaggttc tcccagcttt gagacgtctc 120
agacttacca gttctccaaa acgctatttt ctttaagggt gacacctttt aaaaattagg 180
cacctcaaay atctactgct tttgagcttt tgagttttgc actgtaaaaa gaaaaataca 240
cagtgggatt ttaagtcaaa ttagtttatc taatttttag ggaataattt gaagcatgct 300
ttgtttgcat agattttttt aaaataagct tttccaaatc ataaagagat aagatcttag 360
gtaacatgaa gagactccct tacttattcc taaatmatct atattccaag ggcattttct 420
tatttggaac agttgacctc actgataaag ctgtctc 457
<210> 10
<211> 399
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 203
<223> 99-23444-203 : polymorphic base A or G
<220>
<221> misc_binding
<222> 191. 215
<223> 99-23444-203.probe
<220>
<221> primer_bind
<222> 184..202
<223> 99-23444-203.mis
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
31
<220>
<221> primerbind
<222> 204..222
<223> 99-23444-203.mis complement
<220>
<221> primer_bind
<222> 1..19
<223> 99-23444.pu
<220>
<221> primer_bind
<222> 379..399
<223> 99-23444.rp complement
<400> 10
cttcatagtc aacgaaggct tgaaccaacc tacggatgac tcgtgctttg acccctacac 60
agtttcccat tatgccgttg gagatgagtg ggaacgaatg tctgaatcag gctttaaact 120
gttgtgccag tgcttaggct ttggaagtgg tcatttcaga tgtgattcat ctagtgagta 180
gttgctttgt ccatccactt ccrtgtttgt ctcctcaagt tccatgcatg cactcatgtg 240
ccaaggaagc atgtttggrr aagacacagg ttcttccaaa catgaagcma aacaagagaa 300
tactgtttga ctcgaagtaa twattttgca tcatagaaaa atgatgggaa attttacttg 360
ttggacattg cttcatttca agggttgtat gccaataca 399
<210> 11
<211> 547
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 77
<223> 99-23451-78 : polymorphic base A or G
<220>
<221> misc_binding
<222> 65..89
<223> 99-23451-78.probe
<220>
<221> primer_bind
<222> 58..76
<223> 99-23451-78.mis
<220>
<221> primer_bind
<222> 78..96
<223> 99-23451-78.mis complement
<220>
<221> primer_bind
<222> 1..20
<223> 99-23451.pu
<220>
<221> primer_bind
<222> 529..547
<223> 99-23451.rp complement
<400> 11
ggatatgtaa attgcccccc acaacctttt aaaataagca caatacatct aaaagagctg 60
cacaaaatcc aaagctrttt ataaaattct gtcccaatag tcatctggaa aacttaggtc 120
aacataaagg aattccgttg atataaaatt acaataagat tatttgatgc agaggaaaag 180
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
32
aacagttaga tttattatga tattatattt tcaccacctt agaaactgtg ttagagatga 240
tctccatctt tattccaacg aacaacggtc atgtcttacc ataagtcctg atacaaccac 300
ggatgagctg tcaggagcaa ggttgatttc tttcattggt ccggtcttct ccttgggggt 360
cacccgcact cgatatccag tgagctgaac attgggtggt gtccactggg cgctcaggct 420
tgtgggtgtg acctgagtga acttcaggtc agttggtgca ggaatagctg tcgagattgt 480
cattggttag aggttatctt ataggaaatg ggggaaaagg aaaataaagt gagtycmaag 540
aagtaga 547
<210> 12
<211> 400
<212> DNA
'<213> Homo sapiens
<220>
<221> allele
<222> 306
<223> 99-23452-306 : polymorphic base G or T
<220>
<221> miscbinding
<222> 294..318
<223> 99-23452-306.probe
<220>
<221> primerbind
<222> 287..305
<223> 99-23452-306.mis
<220>
<221> primer_bind
<222> 307..325
<223> 99-23452-306.mis complement
<220>
<221> primer_bind
<222> 1..20
<223> 99-23452.pu
<220>
<221> primer_bind
<222> 380..400
<223> 99-23452.rp complement
<400> 12
tcctctccca attctcaccc aatgaaaaaa tatgttacat cctctatcca ctgcttggtt 60
aaactgaggt tctccataaa aatacttgtt atctatatgc tatgcaatca tctgtgagtt 120
tgagttttga atatgtgcat tgattctctc tcacagtgca gcctgggagc tctattccac 180
cttacaacac cgaggtgact gagaccacca ttgtgatcac atggacgcct gctccaagaa 240
ttggttttaa ggtaaactgc agatgttcct aatctctgtg atacagccct gaagctgtcc 300
ttgtgkttcc catgtagtgg aaacagggtg ctcaggagtc aggagacctg ggttttgtca 360
cctgcttctg tccatacatc tttgactaca ttgtcagggc 400
<210> 13
<211> 450
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 417
<223> 99-28437-417 : polymorphic base C or T
<220>
CA 02368672 2001-09-24
WO 00/56924 PCT/IBOO/00404
33
<221> misc binding
<222> 405._429
<223> 99-28437-417.probe
<220>
<221> primerbind
<222> 398..416
<223> 99-28437-417.mis
<220>
<221> primer_bind
<222> 418..436
<223> 99-28437-417.mis complement
<220>
<221> primer_bind
<222> 1..20
<223> 99-28437.pu
<220>
<221> primer_bind
<222> 431..450
<223> 99-28437.rp complement
<400> 13
gtgaatatta aacatcgacc atctttattt tccaagtaat gtgttatggt tttaagaaaa 60
agtaggaaca cttttgggat agttgcattt ttctggagga aatcttagag gaaaataaat 120
gtcagcctaa tataaaaaac taagtagttt gggcactgtt gtggagaaaa acaccaacac 180
ctattacttt tattaccaat aaaatgaaac ttcatgttca gcattagaat ttttctccct 240
cttttcatca gagtaagcac cactgttttc cttgctgtgc gctctgtgtt tgtacaaagc 300
catttgtaat ggcagaagga gtcttctcca tctgtcccaa cagttccaag cacaagcata 360
acaggatata tttaagaaaa gaactcctcc ctgtattcca gagttttctc tcattcygtg 420
aaatgattaa gatttggata tgatgaaggt 450
<210> 14
<211> 494
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 218
<223> 99-32278-218 polymorphic base A or G
<220>
<221> miscbinding
<222> 206._230
<223> 99-32278-218.probe
<220>
<221> primer_bind
<222> 199..217
<223> 99-32278-218.mis
<220>
<221> primer_bind
<222> 219..237
<223> 99-32278-218.mis complement
<220>
<221> primer_bind
<222> 1..20
<223> 99-32278.pu
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
34
<220>
<221> primerbind
<222> 474..494
<223> 99-32278.rp complement
<220>
<221> allele
<222> 414
<223> 99-32278-414 : polymorphic base C or T
<220>
<221> misc_binding
<222> 402..426
<223> 99-32278-414.probe
<220>
<221> primer_bind
<222> 395..413
<223> 99-32278-414.mis
<220>
<221> primer_bind
<222> 415..433
<223> 99-32278-414.mis complement
<400> 14
gcacttttct atatgcctac ttcattacaa tttcttttaa agataaattt gtgcctggca 60
cagtgtttgg caaaaataag gtataaaaat gtccatgaat ggaaagcact gcgagcttaa 120
agacctgcag ggttctgtgc tctgaggaag ggacataggc tgggctttag aaaggtggcc 180
tggagagaag caggtgtcaa agggcaaggc aacgggarga agaatggagg atccctttca 240
tggactgttt tctccctgtg cccaggggat cccccaatag aaatacactc agtattggtc 300
aggacgttgt tacattatgg atcttctgtc cttctgctgg aaacaacaga cataagatca 360
ttatgcattt cacttaaaca ccagtgaaac tccactcttg acgtttcgaa tgaytgaatt 420
atactagaca tatatatgtt aatggggttc cagtgccaga ccctcccaaa gtgctcaact 480
tccttggtta ctgg 494
<210> 15
<211> 533
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 382
<223> 99-5574-388 : deletion AA
<220>
<221> primer_bind
<222> 1..20
<223> 99-5574.pu
<220>
<221> primer_bind
<222> 513..533
<223> 99-5574.rp complement
<400> 15
ttgacatttg cccagcggag tcatcacctg gaaaccacgg gcagctgaac cctgggaact 60
tgcctcggtg tttataaata cctcagttgc atcaggaccc tacaggtgaa agatcttgat 120
accacacagg tataattaca atctgcaaac ctactcaagg ggagttgcag gtgaagataa 180
ggaagtcagc ctcattccat tacctaatca gattctcagc caaagacaaa cagcaacata 240
tgggacttta aggtgagcag ggagccgaca gcagcgctac tcaaaatgtg gtccgtgatc 300
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
tgcatggctc tcagaattgt ttgttattgg ttcatggcaa gtaaagcaca gaaaatgaga 360
gtaaggattt aaaacatttg gaaaagtttg acaatagttg tgtatctgtt gaaactaatc 420
atttataaat gtgtttctat tttkscatga ctttttcatt ttccattgtt ttgcttcttc 480
agttttatca aagtattggt ctgtaacaaa ttgtgtgtgt tttgttgggg act 533
<210> 16
<211> 472
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 327
<223> 99-5575-330 polymorphic base C or T
<220>
<221> miscbinding
<222> 315..339
<223> 99-5575-330.probe
<220>
<221> primerbind
<222> 308..326
<223> 99-5575-330.mis
<220>
<221> primer_bind
<222> 328..346
<223> 99-5575-330.mis complement
<220>
<221> primer_bind
<222> 1..20
<223> 99-5575.pu
<220>
<221> primer_bind
<222> 452..472
<223> 99-5575.rp complement
<400> 16
gaaaaattgt ttgtgctctg tcacttgtta taagttgtag ctttatctga cacttaccta 60
ccctcaggct tctttttaac tcactctgac ttatttgtct tatccagaga ttgtggattc 120
cctgtcatca aagcagtcta aagggtgtta aaacacctgc ggcccttatt tctttgccat 180
aggctttagg tgacttaaaa aaaaatagta ctgtcctctc actgtctaag gactaccttc 240
cttagtatct ttacataagg aagaaattgg tttttctgtt ttatctgaca aaagagagag 300
atatgaggga gagactgaat tctttcytga aacagaaata ttcctatctc ttatcaagta 360
ttttgattgt ttaatttcct acactaagtc caacaggatt ttataccaaa agcagtagct 420
tccctaaagt ctacttggta gttgttcgtt gccaaagtct attctctctt aa 472
<210> 17
<211> 516
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 354
<223> 99-5582-354 : polymorphic base A or G
<220>
<221> miscbinding
<222> 342._366
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
36
<223> 99-5582-354.probe
<220>
<221> primerbind
<222> 335..353
<223> 99-5582-354.mis
<220>
<221> primer_bind
<222> 355..373
<223> 99-5582-354.mis complement
<220>
<221> primer_bind
<222> 1..19
<223> 99-5582.pu
<220>
<221> primer_bind
<222> 497..516
<223> 99-5582.rp complement
<220>
<221> allele
<222> 71
<223> 99-5582-71 : polymorphic base G or C
<220>
<221> misc_binding
<222> 59..83
<223> 99-5582-71.probe
<220>
<221> primer_bind
<222> 52..70
<223> 99-5582-71.mis
<220>
<221> primer_bind
<222> 72..90
<223> 99-5582-71.mis complement
<400> 17
tcatcagttc aaatagtcct gggcgtgctt tagtttctca tgcttttgag cagagtttta 60
aaataagccc satttgcccc tacagatctc ctgcctggta cagaatatgt agtgagtgtc 120
tccagtgtct acgaacaaca tgagagcaca cctcttagag gaagacagaa aacaggtgag 180
tggtgttggc agtatgacta tccagtagct tttgcctatc aattctgtat aacaaatgaa 240
atgctacttc taaaaataca tctccatttt ttgttgtcat ggtgtgtgta cctttgtcat 300
cacagtatga ttttatcgct ggtctcaaaa actaaaagat accttactca acartcacct 360
agactttcag tcactaacaa attaagaaat ttgttgtctg tccttttaaa aaacattttc 420
taagaagatc tttgttattt agatttagca gacattcctt ttcattaggc agctctgtct 480
aatggctgac ccaacactca ttgtcatcta tttgtc 516
<210> 18
<211> 461
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 424
<223> 99-5590-425 : polymorphic base G or C
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
37
<220>
<221> miscbinding
<222> 412..436
<223> 99-5590-425.probe
<220>
<221> primerbind
<222> 405..423
<223> 99-5590-425.mis
<220>
<221> primer_bind
<222> 425..443
<223> 99-5590-425.mis complement
<220>
<221> primer_bind
<222> 1..19
<223> 99-5590.pu
<220>
<221> primer_bind
<222> 441..461
<223> 99-5590.rp complement
<220>
<221> allele
<222> 99
<223> 99-5590-99 : polymorphic base C or T
<220>
<221> misc_binding
<222> 87..111
<223> 99-5590-99.probe
<220>
<221> primer_bind
<222> 80..98
<223> 99-5590-99.mis
<220>
<221> primer_bind
<222> 100..118
<223> 99-5590-99.mis complement
<400> 18
atgcccattg ratttctacg aattttactt aaactgaaaa tataaataaa gcataatgtt 60
gaccaacaat aactagcata tgatattgaa taaatatayt gttattcaac gttcatttaa 120
caaccagaaa aaaaagaaaa aaattgttat tgttttattg ttctgtttca aacagaaact 180
tcaaactcct agaaaatata atttacagaa attcaatggt ttaaagctaa tgacagaatg 240
ggtggtttac cctctctgtc aatataagac atatatattt ttattcaata tatgaattga 300
cctgtaatca aaaactataa acaagctgta gctaataatc tcagtgatct tttgatgaat 360
gagggatata aagagatgcc ttcagtacaa aaaattcaag ttacaaaagt gtaatactca 420
agastgactt cctgaaacaa gtaagttctc tatgaaaagg a 461
<210> 19
<211> 453
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 379
CA 02368672 2001-09-24
WO 00/56924 PCT/IB00/00404
38
<223> 99-5595-380 : polymorphic base A or G
<220>
<221> misc binding
<222> 367._391
<223> 99-5595-380.probe
<220>
<221> primerbind
<222> 360..378
<223> 99-5595-380.mis
<220>
<221> primer_bind
<222> 380..398
<223> 99-5595-380.mis complement
<220>
<221> primer_bind
<222> 1..18
<223> 99-5595.pu
<220>
<221> primer_bind
<222> 436..453
<223> 99-5595.rp complement
<400> 19
cagatcatgg ttctgaagac cctgtgacac gtcccagttc acctactgtc ttgtgagtca 60
gaatatacaa ataacttttt ggtcctgact ttccccaccc ctacaggatg gtgccatgac 120
aatggtgtga actacaagat tggagagaag tgggaccgtc agggagaaaa tggccagatg 180
atgagctgca catgtcttgg gaacggaaaa ggagaattca agtgtgaccc tcgtatgtca 240
tcacagatca tttttagtgc cttattaagc attctcactt tcattatcag gctgtaactc 300
tcattcacag aaatgattgg agactttagg tctccttgag gagtgaacag tgggtttctt 360
aatcttttga tttgggaarg tggagacaag cttcaaaaat gagtcatgat ttaatgttat 420
tacaggacac tttagcactt gtccaacctg agt 453
<210> 20
<211> 467
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 374
<223> 99-5604-376 : polymorphic base A or G
<220>
<221> misc_binding
<222> 362..386
<223> 99-5604-376.probe
<220>
<221> primer_bind
<222> 355..373
<223> 99-5604-376.mis
<220>
<221> primer_bind
<222> 375..393
<223> 99-5604-376.mis complement
<220>
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<221> primerbind
<222> 1..20
<223> 99-5604.pu
<220>
<221> primerbind
<222> 447..467
<223> 99-5604.rp complement
<400> 20
ctttaccaaa atcactctac ttcagaggga gattaaaaga gatattctga gctagtttca 60
ttctgtgtgt ttgcatacat taatcagatt tagagatgat agccttagct ctgtgccccg 120
gcaaagataa gaactatata atactttttt ttaaacaaaa atttcacaag aattttacag 180
taaaattaga aatagctaaa taataaccta aacatatccc ttaaattaac aagtatatga 240
ggtaagaatg caatcaacat taattggaac ttttattttt gtttaagatt tttctccata 300
ggtttgttga gacttccatg tggttttggc aaaagtaatg ggtatctaaa atttcctgtt 360
attgctatag tacrtcatgc tgtttgaata ttgttaacaa ctatctttat acattttagc 420
attttataaa taattttcaa atatatgtga acaagraatt tagacaa 467
<210> 21
<211> 399
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 135
<223> 99-5605-135 : polymorphic base G or T
<220>
<221> misc_binding
<222> 123..147
<223> 99-5605-135.probe
<220>
<221> primer_bind
<222> 116..134
<223> 99-5605-135.mis
<220>
<221> primer_bind
<222> 136..154
<223> 99-5605-135.mis complement
<220>
<221> primer_bind
<222> 1..18
<223> 99-5605.pu
<220>
<221> primer_bind
<222> 380..399
<223> 99-5605.rp complement
<220>
<221> allele
<222> 90
<223> 99-5605-90 : polymorphic base G or T
<220>
<221> misc_binding
<222> 78..102
<223> 99-5605-90.probe
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WO 00/56924 PCT/IB00/00404
<220>
<221> primerbind
<222> 71..89
<223> 99-5605-90.mis
<220>
<221> primerbind
<222> 91..109
<223> 99-5605-90.mis complement
<400> 21
aagactccag tggctttggg gctctcttgg ttgcccttta tggccacgag ggatacggtg 60
tactcagatg caggctgcag attcctcagk gggtacttgg agacagaggg acccacattg 120
tactgcctgg gctgkcctct tcgggtaagg cccacggtca gtcggtatcc tgttatctgg 180
gcccgaggtg gagtccatct caccaggaca gtagaatcag tttcattgac aaactggagg 240
ttagtgggag catccagttc taggaaaaaa gatgaaacat gccaagaaat atttagatca 300
gtaatgatca taactcaagt cctgaaactt gattgaatgt ctaagttttc tctcctcaag 360
gttgtaacta tgtgaaagtc aaaaccctgr aaaaactga 399
<210> 22
<211> 529
<212> DNA
<213> Homo sapiens
<220>
<221> allele
<222> 323
<223> 99-5608-324 polymorphic base A or G
<220>
<221> misc_binding
<222> 311..335
<223> 99-5608-324.probe
<220>
<221> primer_bind
<222> 304..322
<223> 99-5608-324.mis
<220>
<221> primer_bind
<222> 324..342
<223> 99-5608-324.mis complement
<220>
<221> primer_bind
<222> 1..19
<223> 99-5608.pu
<220>
<221> primer_bind
<222> 509..529
<223> 99-5608.rp complement
<400> 22
caaatcaagt gtagcaaggc aatgtaaaac tttaaaacga tgatatttct ttttaaagtc 60
tgattaacat ttactagttt tacctaattt ttcttgcatt gttgatttct tgcctaaata 120
tagatttttc ttttagtaat gccttttcaa tcttgcccgc ttaaaacaat tctcggggga 180
agcaataacc tgaatcaata aaaacggcaa aagatctttg gaaatagttg gttctcctgt 240
ttgagatcag gagtaaacaa actgtttagc tgggagctta tcaagccatg ctaaaagtgt 300
cagctgacac aaagtaagac gcrttagatt ggggttatca tacaatgggg tttccccaag 360
acaaaactct atactatgct atttgctgag aaatgatcag tacaaaagaa agtttcatca 420
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ttcttgcatt gtgatgctaa aagaaaaggc cttgtaaatg tgtttaattt gctattcgtt 480
hacttcataa atttaatgta tcactttgga gaatccaaca gacattttg 529
<210> 23
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> sequencing oligonucleotide PrimerPU
<400> 23
tgtaaaacga cggccagt 18
<210> 24
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> sequencing oligonucleotide PrimerRP
<400> 24
caggaaacag ctatgacc 18
